Systems, devices, and methods for isotachophoresis

ABSTRACT

The present disclosure relates to fluidic systems and devices for processing, extracting, or purifying one or more analytes. These systems and devices can be used for processing samples and extracting nucleic acids, for example by isotachophoresis. In particular, the systems and related methods can allow for extraction of nucleic acids, including non-crosslinked nucleic acids, from samples such as tissue or cells. The systems and devices can also be used for multiplex parallel sample processing.

CROSS-REFERENCE

This is a divisional application of U.S. patent application Ser. No.16/052,565, filed on Aug. 1, 2018, entitled “Systems, Devices, AndMethods For Isotachophoresis” [Attorney Docket No. 43647-719.201], whichclaims the benefit of U.S. Provisional Application No. 62/540,515, filedAug. 2, 2017, entitled “Isotachophoresis for Purification of NucleicAcids” [Attorney Docket No. 43647-718.101]; U.S. Provisional ApplicationNo. 62/541,086, filed Aug. 3, 2017, entitled “Isotachophoresis Devicesfor Purification of Nucleic Acids” [Attorney Docket No. 43647-719.101];and U.S. Provisional Application No. 62/541,089, filed Aug. 3, 2017,entitled “Nucleic Acid Analysis Using Isotachophoresis and IntercalatingDye” [Attorney Docket No. 43647-720.101], the entire contents of whichare herein incorporated by reference.

This application is related to co-pending PCT Application No.PCT/US2017/015519, filed Jan. 28, 2017, entitled “Isotachophoresis forPurification of Nucleic Acids” [Attorney Docket No. 43647-712.601], theentire contents of which are herein incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder contract number 1R43HG007620-01 awarded by the National Institutesof Health. The government has certain rights in the invention.

BACKGROUND

Formalin-fixed paraffin-embedded (FFPE) samples have been collected,prepared, stored, and archived in large tissue banks for more than acentury. As of 2008, there were over 400 million FFPE samples stored inbiobanks worldwide, and this number is growing. These samples are oftenaccompanied by clinical information such as primary diagnosis,therapeutic regimen, and follow-up data, making them an importantresource for the development of therapeutics and the discovery of genomeand transcriptome biomarkers.

Sample preparation methods to extract and purify nucleic acids from FFPEsamples remain manually intensive and laborious. Approaches for FFPEextraction and purification vary widely but often includedifficult-to-automate and difficult-to-accelerate steps of wax removal,centrifugation, buffer exchanges, temperature control, cross-linkreduction and enzyme treatment. FFPE generally refers to cross-linkingproteins in a sample using formalin and embedding the sample in paraffin(wax). FFPE treatment of a sample often enables the sample to bepreserved over time and can be especially useful for long-term storage.The cross-linked proteins may bind up the DNA and RNA in the sample,thereby generally making it unusable for downstream applications such asamplification, library preparation, or sequencing.

Removal of paraffin and protein crosslinks in FFPE samples may be achallenging process. Deparaffinization is traditionally performed usinghighly flammable xylenes. Alternately or in series, the sample can betreated with other solvents, mineral oil and alkaline chemistry and/orelevated temperature. After deparaffinization, proteins in the samplecan be treated with different agents or subjected to conditions that mayrequire additional time and effort.

At the end of digestion and denaturation, a mix of crosslinked andnon-crosslinked nucleic acids may remain. Removal of the non-crosslinkedmaterial may be important for high quality results from assays such asamplification or sequencing; in some cases, if the fraction ofnon-crosslinked material is too low, the downstream assay may fail toperform resulting in a loss of not only the sample itself, but alsolabor, time and resources.

SUMMARY

Isotachophoresis (ITP) is an electrophoretic technique which can use adiscontinuous buffer containing a leading electrolyte (LE) with a highereffective mobility magnitude and a trailing electrolyte (TE) with alower effective mobility magnitude (e.g., relative to the LE) to focussample species that have a greater effective mobility magnitude than thetrailing electrolyte but a lower effective mobility magnitude than theleading electrolyte. ITP can selectively focus nucleic acids fromsamples by more than 10,000-fold in less than five minutes. The presentdisclosure provides methods and devices employing and automating ITP forsample preparation, including extraction, purification, enrichment, andhighly sensitive quantitation, and is particularly useful for preparingand purifying nucleic acids from FFPE samples and other biologicalsamples.

Sample preparation is important to genomic analysis, yet it remains aprimary source of analysis variability and can require significantmanual labor. The present disclosure includes techniques and devices toaddress this challenge, such as by using on-chip isotachophoresis (ITP)for extraction and purification of nucleic acids. These techniquesinclude methods to enrich (concentrate) non-crosslinked nucleic acids toenable higher yield and higher quality nucleic acid sample preparationand produce more useable samples (e.g., fewer quality-check rejections)from FFPE and other preserved or fresh samples.

The present disclosure includes techniques and devices for automation ofnucleic acid sample preparation from samples, including solid tissue,lysed solid tissue, preserved or fixed tissue samples (e.g., FFPE),whole blood, plasma and serum, buccal swabs, dried blood spots and otherforensic samples, fresh or fresh frozen (FF) tissues, biopsy tissue,organ tissue, solid organ tissue, samples comprising connections (e.g.gap junctions, tight junctions, adherent junctions) between cells,cultured or harvested cells from blood or tissues, stool, and bodilyfluids (e.g., saliva, urine), or any combination thereof. Samples caninclude cellular and cell-free nucleic acids, for both eukaryotic andprokaryotic organisms, or any combination thereof. The techniques of thepresent disclosure, compared to existing approaches, can be faster, lessmanually intensive, more suited for both small and large startingamounts of tissue, and can achieve higher yield from samples and higherquality analyses of samples.

An aspect of the present disclosure provides a fluidic device comprisingan isotachophoresis (ITP) circuit comprising: (a) a first channelcomprising first and second capillary barriers that are spaced apart;and (b) a first loading reservoir in fluid communication with said firstchannel via a first aperture in said first channel, wherein said firstaperture is positioned between said first and second capillary barriersto permit a liquid entering said first channel via said first apertureto flow in one direction along said first channel and arrest at saidfirst capillary barrier and to flow in another direction along saidfirst channel and arrest at said second capillary barrier.

In some embodiments of aspects provided herein, said liquid enteringsaid first channel via said first aperture flows along a path to saidfirst capillary barrier that is longer than a width of said firstchannel. In some embodiments of aspects provided herein, said liquidentering said first channel via said first aperture flows along a pathto said second capillary barrier that is longer than a width of saidfirst channel. In some embodiments of aspects provided herein, saidliquid entering said first channel via said first aperture flows suchthat a meniscus of said first liquid arrests at said first capillarybarrier or at said second capillary barrier. In some embodiments ofaspects provided herein, said first capillary barrier is configured andarranged to be breached by a liquid when a first burst pressure isapplied to said one or more branched fluidic circuits and said secondcapillary barrier is configured and arranged to be breached by saidliquid when a second burst pressure is applied to said one or morebranched fluidic circuits. In some embodiments of aspects providedherein, said first and second burst pressures are about equal. In someembodiments of aspects provided herein, said first burst pressure ishigher than said second burst pressure. In some embodiments of aspectsprovided herein, one or both of said first and second capillary barriersis a cliff capillary barrier. In some embodiments of aspects providedherein, said ITP circuit comprises a second channel in fluidcommunication with said first channel and said first capillary barrieris configured and arranged to arrest flow of a second liquid as it flowsalong said second channel such that a liquid-liquid interface is formedbetween said first and second liquids at said first capillary barrier.In some embodiments of aspects provided herein, one or both of saidfirst and second capillary barriers is a plateau capillary barrier. Insome embodiments of aspects provided herein, said plateau capillarybarrier is configured and arranged so that an air gap forms between saidfirst liquid after said first liquid arrests at said plateau capillarybarrier and a second liquid after said second liquid flows toward saidplateau capillary barrier in another direction and arrests at saidplateau capillary barrier opposite to said first liquid. In someembodiments of aspects provided herein, one or both of said first andsecond capillary barriers comprises a plateau. In some embodiments ofaspects provided herein, one or both of said first and second capillarybarriers comprises a ramp without a plateau. In some embodiments ofaspects provided herein, said first capillary barrier is a cliffcapillary barrier and said second capillary barrier is a plateaucapillary barrier. In some embodiments of aspects provided herein, saidat least one ITP branch further comprises a third capillary barrier thatis a plateau capillary barrier. In some embodiments of aspects providedherein, said first minimum pressure is at least two times higher thansaid second minimum pressure. In some embodiments of aspects providedherein, said fluidic device further comprises a substrate having a firstface and a second face, wherein said first face comprises a plurality ofreservoirs including said first loading reservoir and said second facecomprises a plurality of channels including said first channel, whereinsaid plurality of reservoirs communicate with said plurality of channelsvia through holes in said substrate. In some embodiments of aspectsprovided herein, said ITP circuit further comprises a second loadingreservoir and a second channel, wherein said second loading reservoir isin fluid communication with said second channel via a second apertureand said second channel comprises a third capillary barrier wherein saidthird capillary barrier is configured and arranged to use capillaryforces to arrest a meniscus of a liquid flowing along said secondchannel at said third capillary barrier In some embodiments of aspectsprovided herein, said second channel is adjacent to said secondcapillary barrier within said first channel, and said second capillarybarrier is configured and arranged to use capillary forces to arrest ameniscus of said liquid flowing along said second channel at said secondcapillary barrier. In some embodiments of aspects provided herein, saidITP circuit further comprises a third loading reservoir fluidlyconnected to a third channel via a third aperture, wherein said thirdchannel is fluidly connected to said second reservoir, wherein saidthird channel comprises a fourth capillary barrier positioned betweensaid second aperture and said third aperture. In some embodiments ofaspects provided herein, said first channel or first loading reservoircomprises sample buffer. In some embodiments of aspects provided herein,said second channel or second loading reservoir comprises a firstleading electrolyte buffer. In some embodiments of aspects providedherein, said third channel or third loading reservoir comprises a secondleading electrolyte buffer. In some embodiments of aspects providedherein, the fluidic device further comprises a fourth channel orreservoir in fluidic communication with said first channel and adjacentto said first capillary barrier. In some embodiments of aspects providedherein, said fourth channel or loading reservoir comprises trailingelectrolyte buffer. In some embodiments of aspects provided herein, saidITP circuit comprises an elution channel connected to a first elutionreservoir at an elution junction. In some embodiments of aspectsprovided herein, said elution channel, said first elution reservoir, orboth, comprise a first elution buffer. In some embodiments of aspectsprovided herein, said first aperture, said second aperture, said thirdaperture, said elution junction, or combination thereof, is athrough-hole. In some embodiments of aspects provided herein, said ITPcircuit comprises a second elution reservoir that is separated from saidfirst elution reservoir by said elution channel and wherein said elutionchannel comprises a fifth capillary barrier. In some embodiments ofaspects provided herein, said third, fourth or fifth capillary barrier,in any combination, is a plateau capillary barrier. In some embodimentsof aspects provided herein, said second elution reservoir comprises asecond elution buffer with a higher ion concentration than said firstelution buffer. In some embodiments of aspects provided herein, said ITPcircuit comprises a first leading electrolyte buffer reservoir connectedto a second leading electrolyte buffer reservoir by a buffering channel,wherein said buffering channel comprises first and second leadingelectrolyte buffer that meets at an interface at which a plateaucapillary barrier is situated. In some embodiments of aspects providedherein, said first capillary barrier, said second capillary barrier,said third capillary barrier, said fourth capillary barrier or saidfifth capillary barrier, in any combination, is adjacent to an airchannel comprising a constriction. In some embodiments of aspectsprovided herein, the fluidic device further comprises at least twoadditional ITP circuits, each comprising a first loading reservoir and afirst channel, wherein said first loading reservoir is in fluidcommunication with said first channel via a first aperture and saidfirst channel comprises first and second capillary barriers that arespaced apart and positioned at either side of said first aperture topermit a liquid entering said first channel via said first aperture toflow in one direction along said first channel and arrest at said firstcapillary barrier and to flow in another direction along said firstchannel and arrest at said second capillary barrier. In some embodimentsof aspects provided herein, the fluidic device further comprises atleast five additional ITP circuits, each comprising a first loadingreservoir and a first channel, wherein said first loading reservoir isin fluid communication with said first channel via a first aperture andsaid first channel comprises first and second capillary barriers thatare spaced apart and positioned at either side of said first aperture topermit a liquid entering said first channel via said first aperture toflow in one direction along said first channel and arrest at said firstcapillary barrier and to flow in another direction along said firstchannel and arrest at said second capillary barrier. In some embodimentsof aspects provided herein, said sample reservoir is connected to saidsample channel through a through hole. In some embodiments of aspectsprovided herein, said sample reservoir is closed by a removablematerial. In some embodiments of aspects provided herein, said removablematerial is a film. In some embodiments of aspects provided herein, saidremovable material is a heat-seal material or adhesive material. In someembodiments of aspects provided herein, said removable material is afilm comprising a plastic or a polymer. In some embodiments of aspectsprovided herein, the fluidic device further comprises one or morepneumatic channels opening at one or more pneumatic ports and incommunication with each of said capillary barriers. In some embodimentsof aspects provided herein, the fluidic device further comprises: (a) asubstrate having a first face and a second face, wherein said first facecomprises a plurality of reservoirs including said first loadingreservoir and said second face comprises a plurality of channelsincluding said first channel, wherein said plurality of reservoirscommunicate with said plurality of channels via through holes in saidsubstrate; (b) a layer of material covering said second face, therebyforming closed channels; and (c) a cover covering at least part of saidfirst face and comprising through holes that communicate with ports insaid first face through gaskets. In some embodiments of aspects providedherein, said first face further comprises said one or more pneumaticports. In some embodiments of aspects provided herein, said one or morepneumatic ports have a head height that is shorter than said firstloading reservoir. In some embodiments of aspects provided herein, saidone or more pneumatic ports have a head height that is shorter than atleast one reservoir of said plurality of reservoirs. In some embodimentsof aspects provided herein, said cover layer is attached to said secondface through a solvent heat bond, pressure, adhesive bond, laser weld,or combination thereof. In some embodiments of aspects provided herein,said cover further comprises a porous, air-permeable, hydrophobicmaterial positioned between the through holes in the ports. In someembodiments of aspects provided herein, said first channel is a samplechannel with a depth less than 2 mm. In some embodiments of aspectsprovided herein, said first channel is a sample channel that has a depthgreater than about 10 μm. In some embodiments of aspects providedherein, said sample channel has a depth that is between about 400 μm andabout 1.2 mm. In some embodiments of aspects provided herein, saidsecond channel is a leading electrolyte buffer channel with a depth ofless than about 1 mm. In some embodiments of aspects provided herein,said leading electrolyte buffer channel has a depth that is betweenabout 10 μm and about 600 μm. In some embodiments of aspects providedherein, said elution channel has a depth of less than about 1 mm. Insome embodiments of aspects provided herein, said elution channel has adepth that is between about 10 μm and about 600 μm. In some embodimentsof aspects provided herein, said first, second, or elution channels, ora combination thereof, has a depth of greater than about 40 μm, or adepth greater than about 10 μm. In some embodiments of aspects providedherein, said sample channel has a volume of about 10 μL to about 1 ml.In some embodiments of aspects provided herein, said sample channel,said leading electrolyte buffer channel, said elution channel, orcombination thereof, has a volume of less than about 1 ml. In someembodiments of aspects provided herein, at least one loading reservoircomprises (a) a conical-shaped section in a region of said at least onereservoir bordering said substrate and (b) a cylindrical through-hole oraperture that penetrates through said substrate. In some embodiments ofaspects provided herein, said fluidic device comprises at least oneloading reservoir comprising (a) an entryway for ambient air at one endand (b) an aperture that penetrates said substrate at another end ofsaid loading reservoir, wherein said at least one loading reservoir hasa frustoconical shape with a wider region of said frustoconical shapepositioned at said entryway for ambient air and a narrower regionpositioned at said aperture that penetrates said substrate. In someembodiments of aspects provided herein, said frustoconical shapecomprises a guide wall that is positioned at an angle relative to saidsurface of said substrate within a range of about 60 degrees to about 90degrees. In some embodiments of aspects provided herein, said substratecomprises pneumatic ports configured to have a height or depth tominimize sample loss. In some embodiments of aspects provided herein,said pneumatic ports have a height relative to a surface of saidsubstrate that is shorter than a height of said sample loadingreservoir. In some embodiments of aspects provided herein, saidpneumatic ports on said substrate are inset into a surface of said firstface of said substrate with a depth of from about 1 μm to about 1 mm orare protruding from a surface of said first face of said substrate at aheight of about 0 μm to about 2 mm. In some embodiments of aspectsprovided herein, said pneumatic ports on said substrate are inset into asurface of said first face of said substrate with a depth of from about1 μm to about 500 μm or are protruding from a surface of said first faceof said substrate at a height of about 0 μm to about 1 mm. In someembodiments of aspects provided herein, said first loading reservoir isa sample loading reservoir, said second loading reservoir is a leadingelectrolyte buffer reservoir, said third loading reservoir is a secondleading electrolyte buffer reservoir, said fourth loading reservoir is atrailing electrolyte buffer reservoir, said fifth loading reservoir isan elution reservoir buffer, and said sixth loading reservoir is aelution buffer high reservoir.

An aspect of the present disclosure provides a method of loading saidfluidic device, comprising loading a buffer into said first, second,third, fourth, fifth, or sixth loading reservoirs.

An aspect of the present disclosure provides a method of loading saidfluidic device, comprising loading a buffer into said first, second,third, fourth, fifth, or sixth channels.

In some embodiments of aspects provided herein, said fluidic devicecomprises a first channel comprising a plateau capillary barrieradjacent to a second channel and said loading of said buffer comprisesloading a first buffer into said first channel or reservoir and a secondbuffer into said second channel or reservoir. In some embodiments ofaspects provided herein, said method further comprises applying a firstpositive or negative pneumatic pressure to said fluidic device such thata first and second buffer arrest at a base of a ramp within said plateaucapillary barrier. In some embodiments of aspects provided herein, saidapplying of said first positive or negative pneumatic pressure comprisesincreasing or decreasing said first positive or negative pressure atfixed increments. In some embodiments of aspects provided herein, saidmethod further comprises applying a second positive or negativepneumatic pressure to said fluidic device such that said first andsecond buffers flow long a ramp at either side of said plateau capillarybarrier. In some embodiments of aspects provided herein, said applyingof second positive or negative pneumatic pressure comprises increasingor decreasing said second positive or negative pressure at fixedincrements. In some embodiments of aspects provided herein, said firstand second buffers arrest at a plateau of said plateau capillary barrierwith an air gap between them, said air gap situated above or below saidplateau of said plateau capillary barrier. In some embodiments ofaspects provided herein, said method further comprises applying a thirdpositive or negative pneumatic pressure to said fluidic device such thatsaid first and second liquid enter said air gap such that aliquid-liquid interface forms between said first and second buffer aboveor below said plateau of said plateau capillary barrier.

An aspect of the present disclosure provides a fluidic device comprisinga fluidic channel and disposed in said fluidic channel a capillarybarrier that restricts flow of a liquid in said fluidic channel, whereinsaid capillary barrier comprises: (a) a ramp protruding from a surfaceof said fluidic channel at a first angle; (b) a plateau area, and (c) acliff area extending from said plateau area to said surface of saidfluidic channel and wherein said cliff area intersects with said surfaceat a second angle that is substantially steeper than said first angle.

In some embodiments of aspects provided herein, said second angle is atleast about 10 degrees, at least about 15 degrees, or at least about 20degrees steeper than said first angle. In some embodiments of aspectsprovided herein, said ramp declines or inclines along a length of saidfluidic channel. In some embodiments of aspects provided herein, saidfirst angle is less than 60 degrees. In some embodiments of aspectsprovided herein, said second angle is greater than 60 degrees. In someembodiments of aspects provided herein, said plateau area issubstantially parallel to said surface of said fluidic channel. In someembodiments of aspects provided herein, said plateau area is slanted nomore than about 10 degrees relative to said surface of said fluidicchannel. In some embodiments of aspects provided herein, said ramp,plateau area or cliff area, in any combination, has a substantially flatsurface. In some embodiments of aspects provided herein, said ramp,plateau area or cliff area, in any combination, has a curved surface. Insome embodiments of aspects provided herein, said ramp, plateau area orcliff area, in any combination, has a surface that comprises one or moregrooves, ridges, indentations, steps, etchings, or protrusions. In someembodiments of aspects provided herein, said ramp, plateau area or cliffarea, in any combination, has a surface that comprises regions withfaces at different angles. In some embodiments of aspects providedherein, a width of said ramp, plateau area, or cliff area, substantiallyoccupies a width of said fluidic channel.

An aspect of the present disclosure provides a fluidic device comprisinga fluidic channel and disposed in said fluidic channel a capillarybarrier that restricts flow of a liquid in said fluidic channel, whereinsaid capillary barrier comprises: (a) a first ramp protruding from asurface of said fluidic channel at a first angle that is less than 80degrees; (b) a plateau area; and (c) a second ramp extending from saidplateau area to said surface of said fluidic channel and wherein saidsecond ramp intersects with said surface at a second angle that is lessthan 80 degrees.

In some embodiments of aspects provided herein, said first and secondangles are identical or substantially identical. In some embodiments ofaspects provided herein, said first and second angles are different. Insome embodiments of aspects provided herein, said first ramp, saidsecond ramp, or said plateau area, in any combination, has a surfacethat comprises one or more grooves, ridges, indentations, steps,etchings, or protrusions.

An aspect of the present disclosure provides a fluidic device,comprising a capillary barrier that (a) comprises a cross-sectional areawith a trapezoidal shape; (b) protrudes from an interior surface of saidfluidic channel; (c) has a plateau surface that is substantiallyparallel to said interior surface of said fluidic channel; (d) has aramp surface connecting said plateau surface to said interior surface ofsaid fluidic channel, wherein said ramp surface inclines or declinesalong a length of said fluidic channel; and (e) is configured andarranged to arrest and position a meniscus of a liquid flowing along alength of said fluidic channel.

In some embodiments of aspects provided herein, said capillary barrierextends substantially across a width of said fluidic channel. In someembodiments of aspects provided herein, said capillary barrier isfurther configured and arranged to create a liquid-liquid interface. Insome embodiments of aspects provided herein, said trapezoidal shape isan isosceles trapezoid. In some embodiments of aspects provided herein,said trapezoidal shape is a right trapezoid comprising two angles thatare substantially right angles. In some embodiments of aspects providedherein, said trapezoidal shape is a scalene trapezoid.

In some embodiments of aspects provided herein, said capillary barrieris a “plateau capillary barrier.” In some embodiments of aspectsprovided herein, said capillary barrier is a “cliff capillary barrier.”In some embodiments of aspects provided herein, said fluidic devicecomprises both a cliff capillary barrier and a plateau capillary barrierin the same fluidic circuit. In some embodiments of aspects providedherein, the fluidic device further comprises a sample channel comprisinga cliff capillary barrier. In some embodiments of aspects providedherein, the fluidic device further comprises a plateau capillary barriersituated between buffer channels.

An aspect of the present disclosure provides an isotachophoresis (ITP)system comprising: (a) an interface configured to engage a fluidicdevice, wherein said fluidic device comprises one or more branchedfluidic circuits, each of said branched fluidic circuits comprising aplurality of loading reservoirs including a trailing electrolytereservoir, a first leading electrolyte reservoir and a first elutionbuffer reservoir and wherein said interface comprises: (i) a pneumaticmanifold comprising a plurality of manifold pneumatic channels openingonto one or more manifold ports and communicating with a source ofpositive or negative pneumatic pressure, each manifold port configuredto engage one or more pneumatic ports of said fluidic device when saidfluidic device is engaged with said interface; and (ii) a plurality ofelectrodes, each communicating with a voltage or current source,including a first, second and third electrode, wherein said plurality ofelectrodes are configured to be positioned in said trailing electrolytereservoir, said first leading electrolyte reservoir and said firstelution buffer reservoir, respectively, when said fluidic device isengaged with said interface; (b) a source of positive or negativepneumatic pressure communicating with said pneumatic manifold; and (c) avoltage or current source communicating with said electrodes.

In some embodiments of aspects provided herein, the isotachophoresissystem comprises a motor to engage the interface with an engaged fluidicdevice. In some embodiments of aspects provided herein, said fluidicdevice is engaged with said interface. In some embodiments of aspectsprovided herein, said pneumatic manifold further comprises valves forcontrolling pneumatic pressure to pneumatic channels in at least one ofsaid branched fluidic circuits. In some embodiments of aspects providedherein, the isotachophoresis system further comprises: (d) a ridgehaving a long, narrow tip, a heating element configured to heat saidtip, and an actuator configured to press said ridge tip against afluidic device engaged with said interface to close a plurality offluidic channels in said microfluidic device. In some embodiments ofaspects provided herein, system is configured such that a plurality offluidic channels may be closed with a heat-sealable material, PCR film,parafilm, plastic wrap, adhesive layer, or a material that is notsecured by a seal.

In some embodiments of aspects provided herein, said system isconfigured such that a plurality of fluidic channels within said fluidicdevice may be closed by a load bearing block. In some embodiments ofaspects provided herein, said system is configured such that a pluralityof fluidic channels within said fluidic device may be closed by amechanical actuator block with rubber sealing member.

In some embodiments of aspects provided herein, the system furthercomprises a temperature measuring device. In some embodiments of aspectsprovided herein, the system further comprises a display to displayoperating parameters of the system. In some embodiments of aspectsprovided herein, said display displays temperature.

In some embodiments of aspects provided herein, said display displays ameasure of light detected by a light sensor. In some embodiments ofaspects provided herein, said display displays voltage or current acrossfluidic circuits. In some embodiments of aspects provided herein, saidsystem further comprises a voltage or current measuring device. In someembodiments of aspects provided herein, the system further comprises anoptical assembly comprising one or more light sources configured todirect light to a fluidic channel of said fluidic circuit and one ormore light sensors to detect light emitted from a fluidic channel ofsaid fluidic circuit. In some embodiments of aspects provided herein,said interface further comprises one or more alignment marks foraligning said fluidic device in a particular orientation. In someembodiments of aspects provided herein, the system further comprisessoftware which regulates the electrodes in response to temperature,current or voltage. In some embodiments of aspects provided herein, saidfluidic device further comprises a plurality of branched fluidiccircuits, each of which comprises independent electrical circuitry. Insome embodiments of aspects provided herein, each of said branchedfluidic circuits is coupled to a same voltage or current source or todifferent voltage or current sources.

An aspect of the present disclosure provides a method of creating afluidic circuit comprising: (a) providing a fluidic device, wherein saidfluidic device comprises at least one branched fluidic circuit thatcomprises a trailing electrolyte buffer reservoir, a first channel, afirst leading electrolyte buffer reservoir, a sample loading reservoir,a second leading electrolyte buffer reservoir, and a first elutionbuffer reservoir, all in fluidic communication with another, wherein:(i) said trailing electrolyte buffer reservoir comprises a trailingelectrolyte buffer; (ii) said first leading electrolyte buffer reservoircomprises a first leading electrolyte buffer; (iii) said second leadingelectrolyte buffer reservoir comprises a second electrolyte bufferdifferent from said first electrolyte buffer; and (iv) said firstelution buffer reservoir comprises a first elution buffer; (b) applyingpneumatic pressure to said trailing electrolyte buffer reservoir andsaid leading electrolyte buffer reservoir such that said trailingelectrolyte buffer and said leading electrolyte buffer each enter saidfirst channel and arrest within said first channel with an air gapbetween said trailing electrolyte buffer and said leading electrolytebuffer; (c) loading a sample into said air gap between said trailingelectrolyte buffer and said leading electrolyte buffer within said firstchannel; and (d) applying pneumatic pressure to said second leadingelectrolyte buffer reservoir and said first elution buffer reservoirsuch that said second leading electrolyte buffer and said first elutionbuffer each enter said fluidic circuit virtually simultaneously.

In some embodiments of aspects provided herein, said pneumatic pressureis positive or negative pneumatic pressure. In some embodiments ofaspects provided herein, said applying pneumatic pressure in operation(b) results in said trailing electrolyte buffer being arrested at afirst capillary barrier within said first channel and said leadingelectrolyte buffer being arrested at a second capillary barrier withinsaid first channel. In some embodiments of aspects provided herein, saidapplying pneumatic pressure in operation (d) results in said secondleading electrolyte buffer being arrested at a third capillary barrierwithin said fluidic circuit and said first elution buffer being arrestedat a fourth capillary barrier within said fluidic channel.

In some embodiments of aspects provided herein, said first and secondcapillary barriers are cliff capillary barriers or ramp capillarybarriers. In some embodiments of aspects provided herein, said third andfourth capillary barriers are plateau capillary barriers. In someembodiments of aspects provided herein, said third and fourth capillarybarriers each have a burst pressure that is lower than a burst pressureof said first capillary barrier or of said second capillary barrier. Insome embodiments of aspects provided herein, said sample comprises awetting agent.

An aspect of the present disclosure provides a fluidic device comprisingone or more branched fluidic circuits, wherein each of said branchedfluidic circuits comprises an isotachophoresis (“ITP”) branch and anelution branch in communication with said ITP branch, wherein: (a) saidITP branch comprises a trailing electrolyte buffer reservoir, a samplechannel, a leading electrolyte buffer channel, a first leading bufferelectrolyte reservoir and a second leading electrolyte buffer reservoir,all in communication with each other, wherein: (i) said sample channelis separated from said trailing electrolyte reservoir by a first cliffcapillary barrier and from said leading electrolyte buffer channel by asecond cliff capillary barrier, (ii) said leading electrolyte reservoiris separated from said second leading electrolyte reservoir by a firstplateau capillary barrier; and (b) said elution branch comprises anelution channel, a first elution buffer reservoir and a second elutionbuffer reservoir, all in communication with each other, wherein: (i)said first elution buffer reservoir is separated from said secondelution buffer reservoir a second plateau capillary barrier, and (ii)said leading electrolyte buffer channel is separated from at least partof said elution channel by a third plateau capillary barrier.

An aspect of the present disclosure provides a method of creating afluidic circuit comprising: (a) providing a fluidic device of the anaspect provided herein wherein: (i) said trailing electrolyte bufferreservoir comprises trailing electrolyte buffer; (ii) said first leadingelectrolyte buffer reservoir comprises first leading electrolyte buffer;(iii) said second leading electrolyte buffer reservoir comprises secondleading electrolyte buffer; (iv) said first elution buffer reservoircomprises first elution buffer; and (v) said second elution bufferreservoir comprises second elution buffer; (b) applying negativepneumatic pressure to said first and second cliff capillary barriers toprime trailing electrolyte buffer and first leading electrolyte bufferat said cliff capillary barriers; (c) loading sample into said samplechannel, wherein said sample comprises a wetting agent sufficient tocreate fluidic connections across said first and second cliff capillarybarriers; and (d) applying negative pneumatic pressure to said first,second, and third plateau capillary barriers to create fluidicconnections across said first, second, and third plateau capillarybarriers.

In some embodiments of aspects provided herein, the method furthercomprises: (e) inserting a first electrode into trailing electrolytebuffer in said trailing electrolyte buffer reservoir; (f) inserting asecond electrode into second leading electrolyte buffer in said secondleading electrolyte buffer reservoir; and (g) applying a voltage orcurrent across said first electrode and second electrode.

In some embodiments of aspects provided herein, the method furthercomprises: (h) inserting a third electrode into second elution buffer insaid second elution buffer reservoir; and (i) after operation (g),applying a voltage or current across said first and third electrode,and, optionally, reducing current of said second electrode.

In some embodiments of aspects provided herein, the method furthercomprises adding a topper liquid to said sample reservoir. In someembodiments of aspects provided herein, the method further comprisesspiking said sample with trailing electrolyte buffer. In someembodiments of aspects provided herein, the method further comprisesapplying a voltage or current in response to a triggering event. In someembodiments of aspects provided herein, said voltage is within a rangeof about 0 V to about 1500 V. In some embodiments of aspects providedherein, the method further comprises applying negative pneumaticpressure of between about 0 mpsi and about 200 mpsi. In some embodimentsof aspects provided herein, aid applied negative pneumatic pressure isbetween about 10 mpsi and about 80 mpsi.

An aspect of the present disclosure provides a fluidic device comprisinga fluidic channel, said fluidic channel comprising: (a) a first wallsubstantially parallel to a third wall and a second wall substantiallyparallel to a fourth wall; and (b) a capillary barrier, wherein saidcapillary barrier comprises: (i) a side that is disposed on orintegrated into an interior surface of said second wall and that extendssubstantially between said first wall and said third wall; (ii) firstand second lateral side walls that are connected to, integrated into, oradjacent to said first and third walls respectively, wherein said firstand second lateral side walls each comprise a cross-sectional area witha trapezoidal shape; (iii) a plateau surface that is substantiallyparallel to said second wall and situated between said second and fourthwalls; and (iv) a ramp connecting said second wall to said plateausurface, wherein said ramp inclines or declines along a length of saidfluidic channel.

In some embodiments of aspects provided herein, said trapezoidal shapeis an isosceles trapezoid. In some embodiments of aspects providedherein, said trapezoidal shape is a right trapezoid comprising twoangles that are substantially right angles. In some embodiments ofaspects provided herein, said trapezoidal shape is a scalene trapezoid.In some embodiments of aspects provided herein, said capillary barrieris a “plateau capillary barrier.” In some embodiments of aspectsprovided herein, said capillary barrier is a “cliff capillary barrier.”In some embodiments of aspects provided herein, said fluidic devicecomprises both a cliff capillary barrier and a plateau capillary barrierin the same fluidic circuit. In some embodiments of aspects providedherein, the device further comprises a sample channel comprising a cliffcapillary barrier. In some embodiments of aspects provided herein, thedevice further comprises a plateau capillary barrier situated betweenbuffer channels.

An aspect of the present disclosure provides a fluidic device comprisinga fluidic channel, said fluidic channel comprising a capillary barrierprotruding from a first wall of said fluidic channel into said fluidicchannel, wherein said capillary barrier comprises (i) two lateral sides,each having a cross-sectional area with a trapezoidal shape; (ii) aplateau side substantially parallel to said first wall of said channel;and (iii) a ramp with one edge intersecting said plateau side to form aninterior obtuse angle of said capillary barrier and with an opposingedge intersecting said first wall of said channel to form an interioracute angle of said capillary barrier.

In some embodiments of aspects provided herein, said capillary barrierfurther comprises a side connecting said plateau side to said firstwall. In some embodiments of aspects provided herein, said sideconnecting said plateau side to said first wall is about perpendicularto said first wall. In some embodiments of aspects provided herein, saidside connecting said plateau side to said first wall intersects saidfirst wall at an acute angle. In some embodiments of aspects providedherein, at least one of said lateral sides is substantially parallel to,or integrated into, a second wall of said fluidic channel.

An aspect of the present disclosure provides a fluidic systemcomprising: (a) a first isotachophoresis circuit in a microfluidic chipcomprising: (i) a first sample reservoir; (ii) a trailing electrolytebuffer reservoir comprising trailing electrolyte buffer in fluidcommunication with said sample reservoir; and (iii) a leadingelectrolyte buffer channel comprising leading electrolyte buffer influid communication with said sample reservoir; (b) a sensor configuredto detect a temperature change in said leading electrolyte bufferchannel; and (c) an apparatus configured to monitor voltage or currentin said first isotachophoresis circuit and supply a constant electricalcurrent within said first isotachophoresis circuit.

In some embodiments of aspects provided herein, said leading electrolytechannel comprises an elution channel. In some embodiments of aspectsprovided herein, said sensor is configured and arranged to detect atemperature change in said elution channel. In some embodiments ofaspects provided herein, said fluidic system further comprises anelution well. In some embodiments of aspects provided herein, said firstisotachophoresis circuit further comprises an elution channel comprisingelution buffer.

An aspect of the present disclosure provides a fluidic systemcomprising: (a) a first isotachophoresis circuit in a microfluidic chipcomprising: (i) a first sample reservoir in fluid communication with afirst fluidic channel; (ii) a first, a second, and a third bufferreservoir in fluid communication with said first fluidic channel,wherein said first and second buffer reservoirs are separated by a firstcapillary barrier; and (iii) an elution reservoir in fluid communicationwith said first fluidic channel; (b) a sensor configured to detect atemperature change in said first fluidic channel within said firstisotachophoresis region; and (c) an apparatus configured to monitorvoltage or current and supply a constant electrical current within saidfirst isotachophoresis circuit.

In some embodiments of aspects provided herein, said first fluidicchannel comprising a second capillary barrier adjacent to said firstsample reservoir. In some embodiments of aspects provided herein, saidfirst capillary barrier is a plateau capillary barrier and said secondcapillary barrier is a cliff capillary barrier. In some embodiments ofaspects provided herein, said first capillary barrier is a cliffcapillary barrier, a plateau capillary barrier, or a ramp capillarybarrier. In some embodiments of aspects provided herein, said firstfluidic channel comprises a cliff capillary barrier and a constrictiondownstream of said cliff barrier. In some embodiments of aspectsprovided herein, the system further comprises a temperature sensorconfigured downstream of said constriction.

An aspect of the present disclosure provides a fluidic system, saidfluidic system comprising: a fluidic chip comprising a plurality ofcircuits, wherein each of said circuits comprises an elution channel influid communication with an elution reservoir; and a mechanical membercomprising a ridge, wherein said mechanical member is configured tosimultaneously apply mechanical pressure to a plurality of said elutionchannels via said ridge in order to at least partially close saidelution channels by plastic deformation of at least one wall of saidelution channels.

In some embodiments of aspects provided herein, the system furthercomprises a bottom film bonded to a substrate layer, said bottom layerforming a wall of each of said elution channels, wherein the bottom filmand the substrate layer each comprise materials with the same meltingpoint. In some embodiments of aspects provided herein, each elutionchannel comprises a bend and wherein the ridge at least partially closeseach elution channel in two places across the bend. In some embodimentsof aspects provided herein, said ridge completely closes said channels.

An aspect of the present disclosure provides a method of retrievinganalyte from an assay comprising: introducing said analyte into one ofsaid circuits in said fluidic system of an aspect provided herein;allowing said analyte to migrate to said elution channel in said one ofsaid circuits; and engaging said mechanical member in order to applymechanical pressure to said plurality of elution channels via said ridgein order to at least partially close said elution channels by plasticdeformation of at least one wall of said elution channels.

An aspect of the present disclosure provides a fluidic devicecomprising: a first liquid channel; a gas channel in fluid communicationwith said first liquid channel; a pneumatic port in fluid communicationwith said gas channel; and an air-permeable hydrophobic membranedisposed across said pneumatic port, wherein said hydrophobic membraneis not liquid permeable and is configured to inhibit liquid from exitingsaid pneumatic port when a negative pressure is applied to said gaschannel via said pneumatic port.

In some embodiments of aspects provided herein, the device furthercomprises a gasket disposed over said pneumatic port. In someembodiments of aspects provided herein, the device further comprises aconstriction between liquid and gas channel to inhibit liquid fromexiting said gas channel. In some embodiments of aspects providedherein, said gasket is secured in place by a cover layer comprising achannel communicating with said gas channel through said port. In someembodiments of aspects provided herein, said cover layer comprises aninterference fit configured to maintain a compressive force on saidgasket.

An aspect of the present disclosure provides a method comprising: (a)providing a fluidic circuit comprising: (i) an elution well adjacent toan elution channel that contains elution buffer wherein said elutionchannel is connected to a leading electrolyte channel that containsleading electrolyte buffer, and (ii) a capillary barrier situated at aninterface between said elution buffer and said leading electrolytebuffer; (b) flowing said interface between said leading electrolytebuffer and said elution buffer towards said elution well; and (c)arresting flow of said interface between said leading electrolyte bufferand said elution buffer such that said capillary barrier is fullyengulfed by said leading electrolyte buffer.

In some embodiments of aspects provided herein, the fluidic circuitfurther comprises a sample well in fluidic communication with saidelution well. In some embodiments of aspects provided herein, the methodfurther comprises introducing a nucleic acid sample into said samplewell and applying an electrical current to said fluidic circuit in orderto move said nucleic acid sample over said capillary barrier.

An aspect of the present disclosure provides a method comprising: (a)providing a fluidic device comprising a fluidic circuit having atrailing electrolyte buffer reservoir, a sample channel, a leadingelectrolyte buffer channel and an elution reservoir, all incommunication with each other, wherein: (i) said leading electrolytebuffer channel is fluidly connected to said elution reservoir via anaperture in said leading electrolyte buffer channel situated below saidelution reservoir; (ii) said trailing electrolyte buffer reservoircomprises trailing electrolyte buffer, (ii) said sample channelcomprises an analyte, (iii) said leading electrolyte buffer channelcomprises leading electrolyte buffer, (iv) said elution reservoircomprises elution buffer; and (b) applying a current across said fluidiccircuit to move said analyte to said elution reservoir, wherein saidcurrent is configured and arranged to generate a first temperature at aninterface between said analyte and said trailing electrolyte buffer anda second temperature at an interface between said sample and saidleading electrolyte buffer, wherein a temperature difference existsbetween said first temperature and said second temperature; and wherein,when said analyte reaches said aperture in said leading electrolytebuffer channel situated below said elution reservoir, said analyteenters into said elution reservoir facilitated by said temperaturedifference.

In some embodiments of aspects provided herein, the method furthercomprises pipetting said analyte from said elution reservoir.

An aspect of the present disclosure provides a method of quantifying anucleic acid sample, the method comprising: providing a fluidic devicecomprising an isotachophoresis (ITP) channel comprising a nucleic acidsample, wherein said nucleic acid sample comprises nucleic acidscomplexed with an intercalating dye; performing ITP in said fluidicchannel in order to focus said nucleic acids complexed with saidintercalating dye; and quantifying said nucleic acids complexed withsaid intercalating dye within said channel by measuring intensity ofsaid intercalating dye after ITP has been performed, wherein saidintercalating dye comprises one or more of SYTO™ 13, PicoGreen®,EvaGreen®, or Quantifluor®.

In some embodiments of aspects provided herein, said dye comprises SYTO™13. In some embodiments of aspects provided herein, said dye comprisesPicoGreen®. In some embodiments of aspects provided herein, said dyecomprises EvaGreen®. In some embodiments of aspects provided herein,said dye comprises Quantifluor®. In some embodiments of aspects providedherein, said nucleic acids complexed with said intercalating dye aresituated in a region of said ITP channel that comprise leadingelectrolyte buffer or elution buffer. In some embodiments of aspectsprovided herein, said nucleic acids complexed with said intercalatingdye comprise RNA or DNA, or combination thereof. In some embodiments ofaspects provided herein, said nucleic acid sample further comprises acontaminant. In some embodiments of aspects provided herein, saidcontaminant comprises protein, cellular debris, lipids, plasmamembranes, small molecules, or combination thereof. In some embodimentsof aspects provided herein, said performing ITP in said fluidic channelcause said nucleic acids complexed with said intercalating dye toseparate from said contaminant.

An aspect of the present disclosure provides a fluidic device comprisingone or more branched fluidic circuits, wherein each of said branchedfluidic circuits comprises an isotachophoresis (“ITP”) branch and anelution branch in communication with said ITP branch, wherein: said ITPbranch comprises a trailing electrolyte buffer reservoir, a samplechannel, a leading electrolyte buffer channel, a first leading bufferelectrolyte reservoir and a second leading electrolyte buffer reservoir,all in communication with each other; and said elution branch comprisesan elution channel and an elution well, said elution well comprising afirst and second through-hole in communication with said elutionchannel.

In some embodiments of aspects provided herein, said first and secondthrough-holes are circular. In some embodiments of aspects providedherein, said first through-hole has an elliptical shape. In someembodiments of aspects provided herein, said first through-hole has amaximum dimension across of less than 1.5 mm, or a maximum dimensionacross of less than 1 mm. In some embodiments of aspects providedherein, said second through-hole has a maximum dimension across of lessthan 1.5 mm. In some embodiments of aspects provided herein, said secondthrough-hole has a maximum dimension across of less than 1 mm. In someembodiments of aspects provided herein, said first through-hole and saidsecond through-hole are on a same vertical plane within the elutionwell. In some embodiments of aspects provided herein, said firstthrough-hole and said second through-hole are on a different verticalplane within the elution well. In some embodiments of aspects providedherein, said first through-hole and said second through-hole are alignedwith a longitudinal axis of the elution channel. In some embodiments ofaspects provided herein, said first through-hole is configured toconstrain a pipette tip at a predetermined coupling position. In someembodiments of aspects provided herein, said first through-holecomprises a circular cross-section. In some embodiments of aspectsprovided herein, said first through-hole comprises an ellipticalcross-section. In some embodiments of aspects provided herein, saidfirst through-hole comprises a D-shaped cross-section. In someembodiments of aspects provided herein, said first through-holecomprises a guide wall disposed at an angle within a range of about 60degrees to about 90 degrees relative to the channel. In some embodimentsof aspects provided herein, said elution well comprises a one or morevertical gates separating the first through-hole and the secondthrough-hole. In some embodiments of aspects provided herein, saidelution well comprises a circular cross-section. In some embodiments ofaspects provided herein, said elution well comprises an elongatecross-section.

An aspect of the present disclosure provides a fluidic devicecomprising: a first channel terminating at an end in a first throughhole; a second channel terminating at an end in a second through hole;and a fluid reservoir defined by a wall having a height of no more than25 mm, no more than 15 mm, no more than 10 mm, or greater than 10 mm;wherein the reservoir is in fluidic communication with each of twofluidic channels through the first and second through holes, and whereinthe first through hole enters the reservoir at a position lower in thereservoir than the second through hole.

In some embodiments of aspects provided herein, the first channelcommunicates with a first electrode; and the second channel communicateswith a second electrode; wherein the channels and reservoir comprise anelectrically conductive fluid and wherein application of a voltageacross the first and second electrodes produces a current that travelsthrough the reservoir. In some embodiments of aspects provided herein,said wall has a height within a range of about 8 mm to about 10 mm. Insome embodiments of aspects provided herein, said first and secondthrough holes have areas of about 0.2 mm2 to 7 mm2 and about 0.2 mm2 and7 mm2, respectively. In some embodiments of aspects provided herein,said first and second through holes have areas of about 0.8 mm2 to 1.5mm2 and about 1 mm2 and 2.75 mm2, respectively. In some embodiments ofaspects provided herein, said second through hole enters the reservoirthough a platform in the reservoir positioned about 1 mm to about 6 mmabove a point of entry into said reservoir of said first through hole.In some embodiments of aspects provided herein, said volume of saidreservoir between said first and second through holes is no more thanabout 2.5 ml, 1 ml, or 0.5 ml. In some embodiments of aspects providedherein, said volume of the reservoir between the first and secondthrough holes is 0.1 mL.

An aspect of the present disclosure provides a method comprising:providing any of the fluidic devices described herein, wherein thechannels and reservoir comprise an electrically conductive fluid and thefirst channel further comprises an ionic analyte; applying a voltageacross the first and second electrodes to produce a current that travelsthrough the reservoir; and moving the analyte through the first throughhole into the reservoir.

In some embodiments of aspects provided herein, said analyte comprisesnucleic acid, e.g., RNA or DNA. In some embodiments of aspects providedherein, the method further comprises performing isotachoelectrophoresisin the first channel, whereby the analyte moves into the reservoir.

An aspect of the present disclosure provides a fluidic devicecomprising: a fluidic channel having length and width; a reservoirpositioned above said fluidic channel and fluidically connected to saidfluidic channel through one or a plurality of through holes; wherein atleast part of each through hole is substantially co-extensive with saidfluidic channel across said width of said fluidic channel and has ashape wherein, when said fluidic channel and said reservoir comprise anelectrically conductive fluid and an electric current is passed throughsaid fluidic channel, at least 5%, at least 6%, at least 7%, at least10%, or at least 20% of said electric current passes through saidreservoir.

In some embodiments of aspects provided herein, said reservoir comprisesone through hole. In some embodiments of aspects provided herein, saidreservoir comprises two through holes arranged along a longitudinal axisof the channel. In some embodiments of aspects provided herein, said oneor two through holes have a shape of a square, a rectangle, an oval orhave an elongated dimension in a direction of width. In some embodimentsof aspects provided herein, said one or two through holes comprise oneor two sides that span said width of said fluidic channel, or a width ofsaid reservoir, wherein said sides are each at least 75% linear.

An aspect of the present disclosure provides a method comprising:providing any of the devices described herein, wherein the channel andthe reservoir comprise an electrically conductive fluid and thereservoir comprises an ionic analyte; and passing an electric current ispassed through the channel; wherein the electric current moves at leastsome of the analyte from the reservoir into the channel.

An aspect of the present disclosure provides a fluidic device comprisingone or more branched fluidic circuits, wherein each of said branchedfluidic circuits comprises an isotachophoresis (“ITP”) branch, wherein:said ITP branch comprises a trailing electrolyte buffer reservoir, asample channel, a leading electrolyte buffer channel, a first leadingbuffer electrolyte reservoir and a second leading electrolyte bufferreservoir, all in communication with each other, a sample reservoircomprising a first through-hole in communication with said samplechannel.

In some embodiments of aspects provided herein, said first through-holehas a square or rectangular shape. In some embodiments of aspectsprovided herein, said first through-hole has a maximum dimension withina range of about 0.5 mm to about 5 mm. In some embodiments of aspectsprovided herein, said first through-hole has a maximum dimension ofabout 1.5 mm In some embodiments of aspects provided herein, said firstthrough-hole has a maximum dimension of about 1 mm In some embodimentsof aspects provided herein, said first through-hole has a volume of lessthan about 15 ul. In some embodiments of aspects provided herein, saidfirst through-hole has a volume of about 7 ul. In some embodiments ofaspects provided herein, said first through-hole has a width within arange of about 80% to about 120% of a width of said sample channel. Insome embodiments of aspects provided herein, said first through-hole hasa width of about 100% of the width of said sample channel. In someembodiments of aspects provided herein, the sample reservoir furthercomprises a second through-hole in communication with said samplechannel. In some embodiments of aspects provided herein, the first andsecond through-holes are separated by filler block. In some embodimentsof aspects provided herein, the filler block has a height within thechannel within a range of about 0.2 mm to about 2 mm. In someembodiments of aspects provided herein, the filler block has a heightwithin the channel of about 1.2 mm. In some embodiments of aspectsprovided herein, said second through-hole has a square or rectangularshape. In some embodiments of aspects provided herein, said secondthrough-hole has a maximum dimension within a range of about 0.5 mm toabout 5 mm. In some embodiments of aspects provided herein, said secondthrough-hole has a maximum dimension of about 1.5 mm. In someembodiments of aspects provided herein, said second through-hole has amaximum dimension of about 1 mm. In some embodiments of aspects providedherein, said second through-hole has a volume of less than about 15 ul.In some embodiments of aspects provided herein, said second through-holehas a volume of about 7 ul. In some embodiments of aspects providedherein, said second through-hole has a width within a range of about 80%to about 120% of a width of said sample channel. In some embodiments ofaspects provided herein, said second through-hole has a width within arange of about 80% to about 120% of a width of said sample channel. Insome embodiments of aspects provided herein, when an electric field isapplied to the ITP branch, greater than 10% of an electric currentapplied travels above a top surface of said sample channel across alength of said sample reservoir. In some embodiments of aspects providedherein, said sample reservoir has a conical shape with a narrowerportion having a diameter within a range of about 0.1 mm to about 4 mm.In some embodiments of aspects provided herein, said sample reservoirhas a conical shape with a narrower portion having a diameter within arange of about 1 mm to about 4 mm. In some embodiments of aspectsprovided herein, said sample reservoir has an oval shape.

An aspect of the present disclosure provides a device for performingvertical isotachophoresis, the device comprising: one or morecylindrical columns comprising an interior channel defined by an innerwall of the cylindrical column, each cylindrical column comprising: afirst stage comprising a first gel plug disposed at a first locationwithin the interior channel and a first space disposed within theinterior channel between the first gel plug and an upper end of thecylindrical column; a second stage comprising a second gel plug disposedat a second location within the interior channel, the second locationbeing located below the first location and oriented in line withgravity, and a second space disposed within the interior channel betweenthe first gel plug and the second gel plug; and a third stage comprisinga third gel plug disposed at a third location within the interiorchannel, the third location being located below the second location andoriented in line with gravity, and a third space disposed within theinterior channel between the second gel plug and the third gel plug.

In some embodiments of aspects provided herein, the one or morecylindrical columns comprises a plurality of cylindrical columns, theplurality of cylindrical columns being arranged to conform to a standardmicrotiter plate dimensions. In some embodiments of aspects providedherein, the one or more cylindrical columns has a cross-sectional columnarea of about 9 mm×9 mm. In some embodiments of aspects provided herein,the first space comprises a trailing electrolyte buffer, the secondspace comprises an analyte, and the third space comprises a firstleading electrolyte buffer. In some embodiments of aspects providedherein, the device further comprises a fourth stage comprising a fourthgel plug disposed at a fourth location within the interior channel, thefourth location being located below the third location and oriented inline with gravity, and a fourth space disposed within the interiorchannel between the third gel plug and the fourth gel plug. In someembodiments of aspects provided herein, the fourth space comprises anelution buffer. In some embodiments of aspects provided herein, thedevice further comprises a fifth stage comprising a fifth gel plugdisposed at a fifth location within the interior channel, the fifthlocation being located below the fourth location and oriented in linewith gravity, and a fifth space disposed within the interior channelbetween the fourth gel plug and the fifth gel plug. In some embodimentsof aspects provided herein, the fifth space comprises a second leadingelectrolyte buffer.

An aspect of the present disclosure provides a method for focusing ananalyte, the method comprising: introducing said analyte into saidsecond space of said second stage above said second gel plug of any ofthe vertical or column ITP devices described herein; and applying acurrent across said device to move said analyte in the direction ofgravity from the second space, through the second gel plug, and intosaid third space.

A method of claim 255, further comprising applying said current acrosssaid device to move said analyte in the direction of gravity from thethird space, through the third gel plug, and into said fourth space.

In some embodiments of aspects provided herein, the method furthercomprises removing said analyte from said fourth space.

An aspect of the present disclosure provides a method for samplepurification, comprising: (a) loading into a fluidic device (i) a tissuesample comprising lysed solid tissue, wherein said lysed solid tissuecomprises nucleic acids and a contaminant, (ii) a trailing electrolytebuffer comprising first trailing electrolyte ions with an effectivemobility having a magnitude lower than a magnitude of an effectivemobility of said nucleic acids, and (iii) a leading electrolyte buffercomprising first leading electrolyte ions, with a second effectivemobility, wherein said second effective mobility has a magnitude greaterthan said magnitude of said effective mobility of said nucleic acids;and (b) applying an electric field within said fluidic device to conductisotachophoresis with said first trailing electrolyte ions, said nucleicacids, and said first leading electrolyte ions, thereby purifying saidnucleic acids from said contaminant in said tissue sample.

In some embodiments of aspects provided herein, said effective mobilityof said first trailing electrolyte ions has a magnitude greater than amagnitude of an effective mobility of said contaminant. In someembodiments of aspects provided herein, said fluidic device is amicrofluidic chip and said tissue sample, said trailing electrolytebuffer and said leading electrolyte buffer are loaded into a first zoneof said microfluidic chip. Some embodiments of aspects provided hereinmay further comprise, in said first zone of said microfluidic chip,conducting on said tissue sample at least one sample preparationprocedure selected from the group consisting of (1) removing embeddingmaterial, (2) disrupting tissue, (3) lysing cells, (4) de-crosslinkingsaid nucleic acids, (5) digesting proteins, and (6) digesting saidnucleic acids. In some embodiments of aspects provided herein, saidisotachophoresis is conducted in a second zone of said microfluidicchip, wherein said second zone is separate from and fluidicallyconnected to said first zone. In some embodiments of aspects providedherein, said solid tissue is derived from a solid organ. In someembodiments of aspects provided herein, said lysed solid tissuecomprises a chemical fixative. In some embodiments of aspects providedherein, said chemical fixative is formalin. In some embodiments ofaspects provided herein, said solid tissue is formalin fixed paraffinembedded tissue (FFPE). In some embodiments of aspects provided herein,said lysed solid tissue comprises urea or thiourea. Some embodiments ofaspects provided herein further comprise disrupting cell-cell junctions,extracellular matrix, or connective tissue in order to obtain said lysedsolid tissue. In some embodiments of aspects provided herein, said lysedsolid tissue comprises solid particles. In some embodiments of aspectsprovided herein, said nucleic acids comprise dispersed or solvatednucleic acids. In some embodiments of aspects provided herein, saidcontaminant is selected from the group consisting of crosslinked nucleicacids, embedding material, tissue debris, fixation chemicals, proteins,inhibitors, and combinations thereof. In some embodiments of aspectsprovided herein, said contaminant comprises crosslinked nucleic acids.In some embodiments of aspects provided herein, said tissue sample iscombined with said trailing electrolyte buffer prior to said loading. Insome embodiments of aspects provided herein, said tissue sample iscombined with said leading electrolyte buffer prior to said loading. Insome embodiments of aspects provided herein, said loading of saidleading electrolyte buffer is conducted prior to said loading of saidtissue sample. In some embodiments of aspects provided herein, saidsolid tissue is lysed in said leading electrolyte buffer prior to saidloading of said tissue sample. In some embodiments of aspects providedherein, said solid tissue is lysed in said trailing electrolyte bufferprior to said loading of said tissue sample. In some embodiments ofaspects provided herein, said sample preparation procedure comprises,prior to said applying of said electric field, removing embeddingmaterial by incubating said tissue sample in said fluidic device at atemperature of at least about 37° C. for a duration of at least about 1minute. In some embodiments of aspects provided herein, said temperatureis from about 40° C. to about 80° C. In some embodiments of aspectsprovided herein, said duration is from about 1 minute to about 120minutes. In some embodiments of aspects provided herein, said samplepreparation procedure comprises disrupting tissue or lysing cells byapplying mechanical stress to said tissue sample. In some embodiments ofaspects provided herein, said sample preparation procedure comprisesdisrupting tissue or lysing cells by applying heat to said tissuesample. In some embodiments of aspects provided herein, said applyingheat results in a temperature of said tissue sample from about 30° C. toabout 80° C. In some embodiments of aspects provided herein, said samplepreparation procedure comprises disrupting tissue or lysing cells bycontacting said tissue sample with a solution with pH of at least 10 orby proteolytically digesting said tissue sample. In some embodiments ofaspects provided herein, said proteolytic digestion is conducted at atemperature greater than about 25° C. In some embodiments of aspectsprovided herein, said sample preparation procedure comprises disruptingtissue or lysing cells by applying at least one surfactant to saidtissue sample. In some embodiments of aspects provided herein, saidsample preparation procedure comprises disrupting tissue or lysing cellsby applying a solution comprising urea to said tissue or cell sample. Insome embodiments of aspects provided herein, said solution furthercomprises thiourea. In some embodiments of aspects provided herein, aconcentration of said urea in said solution is within a range of fromabout 4 M to about 9 M and a concentration of said thiourea in saidsolution is in a range of from about 0.5 M to about 3.5 M. In someembodiments of aspects provided herein, a concentration of said urea insaid solution is from about 6.5 M to about 7.5 M and a concentration ofsaid thiourea in said solution is from about 1.5 M to about 2.5 M. Insome embodiments of aspects provided herein, said sample preparationprocedure comprises de-crosslinking said nucleic acids by digestingcrosslinking proteins with proteinase K. In some embodiments of aspectsprovided herein, said sample preparation procedure comprises digestingsaid nucleic acids with DNase or RNase. Some embodiments of aspectsprovided herein further comprise eluting an output solution comprisingsaid purified nucleic acids from an outlet reservoir of said fluidicdevice. In some embodiments of aspects provided herein, a concentrationof said purified nucleic acids in said output solution is at least abouttwo-fold higher than a concentration of said nucleic acids in saidtissue sample. In some embodiments of aspects provided herein, saidtissue sample and said purified nucleic acids in said output solutioncomprise crosslinked nucleic acids and a concentration of saidcrosslinked nucleic acids in said output solution is at least abouttwo-fold lower than a concentration of said crosslinked nucleic acids insaid tissue sample. In some embodiments of aspects provided herein, saidcontaminant is present in said output solution at a concentration thatis at least two-fold less than a concentration of said contaminant insaid tissue sample. In some embodiments of aspects provided herein, saidfirst trailing electrolyte ions comprise caproic acid. In someembodiments of aspects provided herein, said first leading electrolyteions comprise chloride. In some embodiments of aspects provided herein,said trailing electrolyte buffer comprises second trailing electrolyteions having a different effective mobility than said first trailingelectrolyte ions. In some embodiments of aspects provided herein, saidsecond trailing electrolyte ions comprise HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or MOPS(3-(N-morpholino)propanesulfonic acid). In some embodiments of aspectsprovided herein, said second trailing electrolyte ions comprise HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and said firsttrailing electrolyte ions comprise caproic acid. In some embodiments ofaspects provided herein, said second trailing electrolyte ions compriseMOPS (3-(N-morpholino)propanesulfonic acid) and said first trailingelectrolyte ions comprise caproic acid. In some embodiments of aspectsprovided herein, said second trailing electrolyte ions comprise HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and said firsttrailing electrolyte ions comprise MOPS. In some embodiments of aspectsprovided herein, said trailing electrolyte buffer comprises secondtrailing electrolyte ions with a second effective mobility, wherein saidsecond effective mobility has a magnitude about the same as or lowerthan said magnitude of said effective mobility of said contaminant. Insome embodiments of aspects provided herein, said tissue sample loadedinto said fluidic device has a volume of at least 50 μl. Someembodiments of aspects provided herein further comprise, in said firstzone of said microfluidic chip, conducting on said tissue sample a firstsample processing procedure, and in a second zone of said microfluidicchip, conducting on said tissue sample an enzymatic reaction. In someembodiments of aspects provided herein, said first sample processingprocedure comprises removal of embedding material, disruption of tissue,or cell lysis, and said enzymatic reaction comprises de-crosslinkingsaid nucleic acids, digesting proteins, or digesting nucleic acids. Insome embodiments of aspects provided herein, said first zone and saidsecond zone each are each heated to a temperature above 37° C. In someembodiments of aspects provided herein, said first zone is heated to atemperature of about 60° C. to 100° C. during said first sampleprocessing procedure and wherein said second zone is heated to atemperature of 40° C. to 60° C.

An aspect of the present disclosure provides a method for simultaneouslypurifying nucleic acids from at least two different samples comprising:(a) loading into a first channel of a microfluidic chip (i) a firstsample comprising first nucleic acids and a first contaminant, (ii) afirst trailing electrolyte buffer comprising first trailing ions,wherein a magnitude of an effective mobility of said first trailing ionsis less than a magnitude of an effective mobility of said first nucleicacids, and (iii) a first leading electrolyte buffer comprising firstleading ions, wherein a magnitude of an effective mobility of said firstleading ions is greater than said magnitude of said effective mobilityof said first nucleic acids; (b) loading into a second channel of saidmicrofluidic chip (i) a second sample comprising second nucleic acidsand a second contaminant, (ii) a second trailing electrolyte buffercomprising second trailing ions, wherein a magnitude of said secondtrailing ions is less than a magnitude of an effective mobility of saidsecond nucleic acids, and (iii) a second leading electrolyte buffercomprising second leading ions, wherein a magnitude of an effectivemobility of said second leading ions is greater than said magnitude ofsaid effective mobility of said second nucleic acids; and (c) applying afirst electric field within said microfluidic chip to conductisotachophoresis in said first channel with said first trailing ions,said first nucleic acids, and said first leading ions, and applying asecond electric field to conduct isotachophoresis in said second channelwith said second trailing ions, said second nucleic acids, and saidsecond leading ions, thereby simultaneously purifying said first nucleicacids from said first contaminant and said second nucleic acids fromsaid second contaminant.

In some embodiments of aspects provided herein, said first sample andsaid second sample are different sample types. In some embodiments ofaspects provided herein, said first nucleic acids and said secondnucleic acids are different types or lengths of nucleic acids. In someembodiments of aspects provided herein, said first trailing electrolytebuffer or said first leading electrolyte buffer further comprises alysis agent or a tissue disruption agent. In some embodiments of aspectsprovided herein, said lysis agent or said tissue disruption agentcomprises one or more agents selected from the group consisting of asolution with pH greater than about 12, a proteinase, urea, thiourea,and a surfactant. In some embodiments of aspects provided herein, saidfirst sample comprises lysed solid tissue. In some embodiments ofaspects provided herein, said second sample comprises lysed cells. Insome embodiments of aspects provided herein, said first sample does notcontact said second sample during said conducting of isotachophoresis.Some embodiments of aspects provided herein further comprise loadinginto a third channel of said microfluidic chip (i) a third samplecomprising third nucleic acids and a third contaminant, (ii) a thirdtrailing electrolyte buffer comprising third trailing ions, wherein amagnitude of an effective mobility of said third trailing ions is lessthan a magnitude of an effective mobility of said third nucleic acids,and (iii) a third leading electrolyte buffer comprising third leadingions, wherein a magnitude of an effective mobility of said third leadingions is greater than said magnitude of said effective mobility of saidthird nucleic acids, wherein said electric field is applied within saidmicrofluidic chip to conduct said isotachophoresis in said third channelwith said third trailing ions, said third nucleic acids, and said thirdleading ions, thereby simultaneously purifying said first nucleic acidsfrom said first contaminant, said second nucleic acids from said secondcontaminant and said third nucleic acids from said third contaminant. Insome embodiments of aspects provided herein, said first and secondelectric fields are generated from a single electrode pair. In someembodiments of aspects provided herein, said first and second electricfields are generated from different electrode pairs. In some embodimentsof aspects provided herein, said first and second channels are coupledto independent sensors. In some embodiments of aspects provided herein,feedback from said independent sensors is used to independently controlsaid first and second electric fields. In some embodiments of aspectsprovided herein, said independent sensors sense voltage and saidfeedback is used to control current (or resistance) within said firstand second channels. In some embodiments of aspects provided herein,said nucleic acids comprise DNA. In some embodiments of aspects providedherein, said nucleic acids comprise RNA.

As aspect of the present disclosure provides a method for samplepurification, comprising: (a) loading onto a fluidic device (i) a samplecomprising fixed cells, fixed tissue, or embedded tissue, wherein saidsample comprise nucleic acids, (ii) a trailing electrolyte buffercomprising trailing electrolytes, wherein said trailing electrolyteshave a lower effective mobility than said nucleic acids, and (iii) aleading electrolyte buffer comprising leading electrolytes, wherein saidleading electrolytes have a higher effective mobility than said nucleicacids; and (b) applying an electric field on said fluidic device toconduct isotachophoresis with said trailing electrolytes, said nucleicacids, and said leading electrolytes, thereby purifying said nucleicacids from a contaminant in said sample.

In some embodiments of aspects provided herein, said contaminant isselected from the group consisting of crosslinked nucleic acids,embedding material, fixation chemicals, enzymes, and inhibitors. In someembodiments of aspects provided herein, said sample comprises said fixedcells, said fixed tissue, or both said fixed cells and said fixedtissue. In some embodiments of aspects provided herein, said sample isformalin-fixed. In some embodiments of aspects provided herein, saidsample comprises said embedded tissue. In some embodiments of aspectsprovided herein, said sample comprises said tissue embedded in paraffin.In some embodiments of aspects provided herein, said sample is aformalin-fixed paraffin-embedded (FFPE) tissue sample. In someembodiments of aspects provided herein, said sample comprises a tissuebiopsy. In some embodiments of aspects provided herein, said sample is adissected formalin-fixed paraffin-embedded (FFPE) sample. Someembodiments of aspects provided herein further comprise comparing acharacteristic of said nucleic acids to nucleic acids from othersamples, wherein said characteristic is an expression level, a nucleicacid sequence, a molecular weight, nucleic acid integrity, nucleic-acidstranded-ness (e.g. double- versus single-stranded), or nucleic acidpurity. In some embodiments of aspects provided herein, said sample is atumor sample. In some embodiments of aspects provided herein, saidtrailing electrolyte buffer has a pH of greater than about 7. Someembodiments of aspects provided herein further comprises, prior to saidapplying said electric field, incubating said tissue sample in saidfluidic device at a temperature of at least about 37° C. for a durationof at least about 1 minute. In some embodiments of aspects providedherein, said temperature is from about 40° C. to about 80° C. In someembodiments of aspects provided herein, said duration is from about 1minute to about 120 minutes. In some embodiments of aspects providedherein, said leading electrolyte buffer comprises proteinase K. Someembodiments of aspects provided herein further comprise removing proteincrosslinks from said nucleic acids using said proteinase K. Someembodiments of aspects provided herein further comprise, after saidapplying said electric field, removing protein crosslinks from saidnucleic acids using heat. Some embodiments of aspects provided hereinfurther comprise eluting an output solution comprising said purifiednucleic acids from an outlet reservoir of said fluidic device. In someembodiments of aspects provided herein, a concentration of said purifiednucleic acids in said output solution is at least about two-fold higherthan a concentration of said nucleic acids in said tissue sample. Insome embodiments of aspects provided herein, a concentration of saidcrosslinked nucleic acids in said output solution is at least abouttwo-fold lower than a concentration of said crosslinked nucleic acids insaid tissue sample. In some embodiments of aspects provided herein, saidoutput solution has a volume equal to or less than about 50 μL. In someembodiments of aspects provided herein, said tissue sample has a mass ofat least about 1 ng. In some embodiments of aspects provided herein,said tissue sample has a volume greater than 25 μL. In some embodimentsof aspects provided herein, said trailing electrolytes have a highereffective mobility than said contaminant. In some embodiments of aspectsprovided herein, said trailing electrolytes comprise (i) first ions,wherein said first ions have a higher effective mobility magnitude thansaid contaminant, and (ii) second ions, wherein said second ions have aneffective mobility magnitude about the same as or lower than saidcontaminant. In some embodiments of aspects provided herein, saidconducting isotachophoresis quenches a pH of said tissue sample to about7.5. Some embodiments of aspects provided herein further comprise, priorto said loading, conducting de-paraffinization on said sample. Someembodiments of aspects provided herein further comprise detecting aconcentration of said nucleic acids. In some embodiments of aspectsprovided herein, said concentration is less than or equal to about 1picogram per microliter (pg/μL).

An aspect of the present disclosure provides a method for samplepurification, comprising: (a) loading into a fluidic device (i) a tissuesample comprising lysed solid tissue and nucleic acids, (ii) a trailingelectrolyte buffer, said trailing electrolyte buffer comprising trailingelectrolyte ions with a first effective mobility, wherein said firsteffective mobility has a magnitude lower than a magnitude of aneffective mobility of said nucleic acids, (iii) a first leadingelectrolyte buffer in a first leading electrolyte reservoir, said firstleading electrolyte buffer comprising first leading electrolyte ionswith a second effective mobility, wherein said second effective mobilityhas a magnitude greater than said magnitude of said effective mobilityof said nucleic acids, and (iv) a second leading electrolyte buffer in asecond leading electrolyte reservoir, said second leading electrolytebuffer comprising second leading electrolyte ions with a third effectivemobility, wherein said third effective mobility has a magnitude greaterthan said magnitude of said effective mobility of said nucleic acids,wherein said first leading electrolyte buffer is different from saidsecond leading electrolyte buffer; (b) first conducting isotachophoresiswith said trailing electrolyte ions, said nucleic acids, and said firstleading electrolyte ions, thereby purifying said nucleic acids from saidcontaminant in said tissue sample; and (c) second conductingisotachophoresis with said trailing electrolyte ions, said nucleicacids, and said second leading electrolyte ions.

In some embodiments of aspects provided herein, said second conductingisotachophoresis comprises changing an applied current from a firstchannel to a second channel. In some embodiments of aspects providedherein, said first leading electrolyte ions are the same as said secondleading electrolyte ions, and wherein a concentration of said firstleading electrolyte ions in said first leading electrolyte buffer isdifferent from a concentration of said second leading electrolyte ionsin said second leading electrolyte buffer. In some embodiments ofaspects provided herein, said second effective mobility has a magnitudegreater than said magnitude of said third effective mobility. In someembodiments of aspects provided herein, said first leading electrolyteions are different from said second leading electrolyte ions. In someembodiments of aspects provided herein, said first leading electrolyteions are the same as said second leading electrolyte ions, and wherein aconcentration of said first leading electrolyte ions in said firstleading electrolyte buffer is the same as a concentration of said secondleading electrolyte ions in said second leading electrolyte buffer, andwherein said first leading electrolyte buffer comprises third leadingelectrolyte ions. In some embodiments of aspects provided herein, saidfirst leading electrolyte ions are the same as said second leadingelectrolyte ions, and wherein a concentration of said first leadingelectrolyte ions in said first leading electrolyte buffer is the same asa concentration of said second leading electrolyte ions in said secondleading electrolyte buffer, and wherein said second leading electrolytebuffer comprises third leading electrolyte ions. Some embodiments ofaspects provided herein further comprise collecting said nucleic acidsin said second leading electrolyte reservoir and removing said nucleicacids from said second leading electrolyte reservoir. In someembodiments of aspects provided herein, said first conductingisotachophoresis and said second conducting isotachophoresis areperformed by applying a single electric field. In some embodiments ofaspects provided herein, said first conducting isotachophoresis and saidsecond conducting isotachophoresis are performed by applying more thanone electric field. In some embodiments of aspects provided herein, theconcentration of said second leading electrolyte ions in said secondleading electrolyte buffer is less than 50 mM. In some embodiments ofaspects provided herein, said second leading electrolyte buffercomprises 50 mM Tris HCl.

An aspect of the present disclosure provides a microfluidic devicecomprising: (a) a first isotachophoresis region in a microfluidic chipcomprising: (i) a first sample reservoir in fluid communication with afirst fluidic channel, (ii) a first buffer reservoir in fluidcommunication with said first fluidic channel, and (iii) a second bufferreservoir in fluid communication with said first channel; and (b) asecond isotachophoresis region in said microfluidic chip comprising: (i)a second sample reservoir in fluid communication with a second fluidicchannel, (ii) a third buffer reservoir in fluid communication with saidsecond fluidic channel, and (iii) a fourth buffer reservoir in fluidcommunication with said second channel, wherein said firstisotachophoresis region is not in fluid communication with said secondisotachophoresis region and wherein said microfluidic device isconfigured to independently control a first electric circuit thatapplies current to said first isotachophoresis region and a secondelectric circuit that applies current to said second isotachophoresisregion.

In some embodiments of aspects provided herein, a leakage rate betweensaid first and second isotachophoresis regions is less than 1 μl perhour. In some embodiments of aspects provided herein, current leakagebetween said first and second isotachophoresis regions is less than 1μA. In some embodiments of aspects provided herein, an impedance isgreater than 1 megaOhm. In some embodiments of aspects provided herein,said first fluidic channel holds a liquid volume greater than 100 μl. Insome embodiments of aspects provided herein, said first fluidic channelis separated from said second fluidic channel by a distance that is atleast 5-fold less than a width of said first channel. In someembodiments of aspects provided herein, said microfluidic device isconfigured to control said first electric circuit simultaneously withsaid second electric circuit. Some embodiments of aspects providedherein further comprise an elution reservoir in fluid communication tosaid first channel, wherein a temperature sensor is situated within 5 mmof said elution reservoir.

An aspect of the present disclosure provides a method, comprising: (a)providing an electrokinetic fluidic device comprising a sample inputreservoir in fluid communication with a channel; (b) loading a samplevolume into said sample input reservoir; (c) moving at least 50% of saidsample volume from said sample input reservoir to said channel, withoutadding additional volume to said sample input reservoir; and (d)applying an ionic current through said channel.

In some embodiments of aspects provided herein, said moving is conductedwith aid of gravity. In some embodiments of aspects provided herein,said ionic current does not substantially pass through said channel. Insome embodiments of aspects provided herein, said at least 50% of saidsample volume comprises at least 80% of said sample volume. In someembodiments of aspects provided herein, said sample volume comprisesnucleic acids. In some embodiments of aspects provided herein, saidsample volume comprises a tissue sample or a formalin-fixedparaffin-embedded (FFPE) sample. In some embodiments of aspects providedherein, said applying an ionic current comprises conductingisotachophoresis. In some embodiments of aspects provided herein, atotal sample volume loaded into said sample input reservoir is less thanor equal to an internal volume of said input reservoir. In someembodiments of aspects provided herein, said sample input reservoircomprises a top region connected to a bottom region via a taperedregion, wherein said top region has a first diameter and said bottomregion has a second diameter, wherein said first diameter is at leasttwo-fold longer than said second diameter in order to facilitate saidmoving at least 50% of said sample volume from said sample inputreservoir to said channel. In some embodiments of aspects providedherein, said sample volume is at least 25 μl. In some embodiments ofaspects provided herein, said sample volume is at least 50 μl. In someembodiments of aspects provided herein, said sample volume is at least100 μl.

An aspect of the present disclosure provides a microfluidic chipcomprising: a first sample input reservoir, wherein said first sampleinput reservoir comprises a top region connected to a bottom region viaa tapered region, wherein said top region has a first inner hydraulicdiameter and said bottom region has a second inner hydraulic diameter,wherein said first inner hydraulic diameter is at least 2-fold longerthan said second inner hydraulic diameter and wherein said first sampleinput reservoir is in fluid communication with a first channel; a firstbuffer reservoir in fluid communication with said first channel, whereinsaid first sample reservoir is configured so that a free surface of aliquid in said first sample reservoir has a negligible buffer headheight difference relative to a liquid in said first buffer reservoir;and a second buffer reservoir in fluid communication with said firstchannel.

In some embodiments of aspects provided herein, said first innerhydraulic diameter is a range of about 1 mm to about 15 mm. In someembodiments of aspects provided herein, said second inner hydraulicdiameter is a range of about 0.5 mm to about 5 mm. In some embodimentsof aspects provided herein, said first sample reservoir is configured tohold a sample volume of at least 100 μl. In some embodiments of aspectsprovided herein, said microfluidic chip is configured to move at least50% of said sample volume from said first sample reservoir to said firstchannel when a vacuum is applied thereto. In some embodiments of aspectsprovided herein, said microfluidic chip is configured to conductisotachophoresis on a sample that enters said first channel.

An aspect of the present disclosure provides a method of extractingnucleic acids, comprising: (a) exposing a biological sample comprisingcells or tissue to a solution comprising urea or thiourea, therebylysing said cells or tissue within said biological sample and producinga cellular lysate; (b) introducing said cellular lysate into a device;and (c) performing isotachophoresis with said device in order to isolatenucleic acids from said cellular lysate.

Some embodiments of aspects provided herein further comprise digestingsaid sample with proteinase K. In some embodiments of aspects providedherein, said solution comprises urea and thiourea. In some embodimentsof aspects provided herein, said solution comprises a ratio of urea tothiourea of about 2 to 1. In some embodiments of aspects providedherein, a concentration of said urea in said solution is from about 4 Mto about 9 M and a concentration of said thiourea in said solution isfrom about 0.5 M to about 3.5 M. In some embodiments of aspects providedherein, a concentration of said urea in said solution is from about 6.5M to about 7.5 M and a concentration of said thiourea in said solutionis from about 1.5 M to about 2.5 M. In some embodiments of aspectsprovided herein, said solution comprises trailing electrolyte ions orleading electrolyte ions or both trailing electrolyte ions and leadingelectrolyte ions.

An aspect of the present disclosure provides a method of purifying highmolecular weight nucleic acids from a tissue sample, comprising: (a)loading into a fluidic device: (i) a cellular sample comprising genomicDNA and a contaminant, wherein said cellular sample is contacted with alysis buffer prior to or after said loading of said cellular sample intosaid fluidic device, (ii) a trailing electrolyte buffer, said trailingelectrolyte buffer comprising trailing electrolyte ions with a firsteffective mobility, wherein said first effective mobility has amagnitude lower than a magnitude of an effective mobility of said highmolecular weight nucleic acids and a magnitude greater than a magnitudeof said contaminant, and (iii) a first leading electrolyte buffer, saidfirst leading electrolyte buffer comprising first leading electrolyteions with a second effective mobility, wherein said second effectivemobility has a magnitude greater than said magnitude of said effectivemobility of said high molecular weight nucleic acids; (b) conductingisotachophoresis with said trailing electrolyte ions, said highmolecular weight nucleic acids, and said first leading electrolyte ions,thereby separating said high molecular weight nucleic acids from saidcontaminant and enriching said high molecular weight nucleic acids in anisotachophoresis zone; and (c) eluting said genomic DNA into a solutionin an output reservoir, wherein greater than 50% of the mass of nucleicacids within said solution are greater than 30 kilobases.

In some embodiments of aspects provided herein, said lysis buffer doesnot comprise an alkaline buffer. In some embodiments of aspects providedherein, said lysis buffer comprises octylphenol ethoxylate. In someembodiments of aspects provided herein, greater than 50% of the mass ofnucleic acids within said solution are greater than 50 kilobases.

An aspect of the present disclosure provides a method of conductingisotachophoresis, comprising: (a) providing a fluidic device comprisinga first channel in fluid communication with a sample input reservoircomprising a tissue sample comprising lysed solid tissue, a first bufferreservoir comprising a first leading electrolyte buffer, and a secondbuffer reservoir comprising a trailing electrolyte buffer; (b)contacting a first electrode to said first leading electrolyte buffer insaid first buffer reservoir; (c) contacting a second electrode to saidtrailing electrolyte buffer in said second buffer reservoir; and (d)applying an electric field within said fluidic device to conductisotachophoresis, wherein said isotachophoresis occurs without directcontact between said tissue sample and said first and second electrodes.

In some embodiments of aspects provided herein, said fluidic devicefurther comprises a third buffer reservoir in fluid communication withsaid first channel and said first buffer reservoir, said third bufferreservoir comprising a lower concentration of said first leadingelectrolyte buffer than said first buffer reservoir. In some embodimentsof aspects provided herein, said third buffer reservoir and said firstbuffer reservoir are connected by a second channel comprising one ormore capillary barriers to limit pressure-driven flow within said secondchannel and between said third buffer reservoir and said first bufferreservoir. In some embodiments of aspects provided herein, said fluidicdevice further comprises an elution reservoir. In some embodiments ofaspects provided herein, said elution reservoir is in fluidcommunication with a fourth buffer reservoir.

An aspect of the present disclosure provides a microfluidic system, saidmicrofluidic system comprising: (a) a microfluidic chip comprising afirst channel and a first reservoir in fluid communication with saidfirst channel, wherein said first channel and said first reservoir meetat a first junction; and (b) a mechanical member comprising a firsttooth, wherein said mechanical member is configured to apply mechanicalpressure to said first channel via said first tooth in order to at leastpartially close said first channel by plastic deformation of at leastone wall of said first channel and increase fluid resistance betweensaid first channel and said first reservoir.

In some embodiments of aspects provided herein, said microfluidic chipfurther comprises a second reservoir in fluid communication with saidfirst reservoir and a second channel connecting said first reservoir andsaid second reservoir, and wherein said mechanical member furthercomprises a second tooth configured to apply mechanical pressure to saidsecond channel in order to plastically close said second channel andprevent fluid communication between said first reservoir and said secondreservoir. In some embodiments of aspects provided herein, said firsttooth is configured to deliver mechanical pressure to said firstjunction in order to close said first channel by plastic deformation ofat least one wall of said first channel. In some embodiments of aspectsprovided herein, said first tooth is configured to heat said firstchannel. In some embodiments of aspects provided herein, said mechanicalmember comprises a material with a Young's modulus of elasticity greaterthan a Young's modulus of elasticity of said first channel. In someembodiments of aspects provided herein, said microfluidic system isconfigured to perform isotachophoresis. In some embodiments of aspectsprovided herein, said first tooth is thermally coupled to a heatingelement. In some embodiments of aspects provided herein, said firsttooth is heated to a temperature greater than the glass transitiontemperature of said at least one wall of said first channel. Someembodiments of aspects provided herein comprise a method of completing aprocess in a fluidic system comprising using said microfluidic system toat least partially close said first channel by plastic deformation,thereby increasing resistance to fluid flow between said first channeland said first reservoir. In some embodiments of aspects providedherein, said first tooth of said mechanical member applies a force of atleast 0.25 lbs to said first channel. In some embodiments of aspectsprovided herein, said process in said fluidic system isisotachophoresis.

An aspect of the present disclosure provides a method of performingisotachophoresis on a sample comprising nucleic acids comprising: (a)loading said sample comprising nucleic acids into a first reservoir of amicrofluidic chip; (b) loading a trailing electrolyte buffer into asecond reservoir of said microfluidic chip, wherein said trailingelectrolyte buffer comprises first trailing electrolyte ions with aneffective mobility having a magnitude lower than a magnitude of aneffective mobility of said nucleic acids; (c) loading a leadingelectrolyte buffer into a third reservoir of said microfluidic chip,wherein said third reservoir comprises first leading electrolyte ionswith a second effective mobility, wherein said second effective mobilityhas a magnitude greater than said magnitude of said effective mobilityof said nucleic acids; (d) applying an electric field within saidmicrofluidic chip to conduct isotachophoresis with said first trailingelectrolyte ions, said nucleic acids, and said first leading electrolyteions, thereby confining said nucleic acids, or a portion thereof, to anisotachophoresis zone; and (e) using a temperature sensor to sense atemperature change in or near said isotachophoresis zone, whereinfeedback from said temperature sensor is used to control said electricfield.

In some embodiments of aspects provided herein, said control of saidelectric field results in positioning of said nucleic acids, or portionthereof, in an elution reservoir or region of said microfluidic chip. Insome embodiments of aspects provided herein, said temperature sensor islocated within at most 8 mm of said elution reservoir. In someembodiments of aspects provided herein, said temperature change iswithin a range of about 0.2° C. to 5° C. In some embodiments of aspectsprovided herein, said applied electric field causes said leadingelectrolyte and said trailing electrolyte to meet at an isotachophoresisinterface and said temperature sensor senses said isotachophoresisinterface.

An aspect of the present disclosure provides a microfluidic devicecomprising: (a) a first isotachophoresis region in a microfluidic chipcomprising: (i) a first sample reservoir in fluid communication with afirst fluidic channel; (ii) a first, a second, and a third bufferreservoir in fluid communication with said first fluidic channel,wherein said first and second buffer reservoirs are separated by acapillary barrier; and (iii) an elution reservoir in fluid communicationwith said first fluidic channel; (b) a sensor configured to detect atemperature change in said first fluidic channel within said firstisotachophoresis region; and (c) an apparatus positioned to supplyelectrical current within said first channel within said firstisotachophoresis region.

Some embodiments of aspects provided herein further comprise acontroller configured to trigger a reduction or elimination of saidelectrical current when said sensor receives a thermal signal. In someembodiments of aspects provided herein, said temperature change is anincrease in temperature within a range of about 0.2° C. to 5° C. In someembodiments of aspects provided herein, said microfluidic device isfurther configured to isolate a sample of nucleic acids in said elutionreservoir after said sensor detects a change in temperature. In someembodiments of aspects provided herein, said sensing of said nucleicacids is performed with a sensor located within at most 8 mm of saidelution reservoir. In some embodiments of aspects provided herein, saidfirst channel comprises a single sensor.

An aspect of the present disclosure provides a kit comprising: (a) saidmicrofluidic device of claim 111, said microfluidic device of claim 165,or said microfluidic chip of claim 128; (b) a trailing electrolytebuffer comprising trailing electrolytes; and (c) a leading electrolytebuffer comprising leading electrolytes.

In some embodiments of aspects provided herein, said trailingelectrolyte buffer comprises a mixture of at least two electrolytes withdifferent effective mobilities. In some embodiments of aspects providedherein, said mixture comprises (i) a first electrolyte that has a lowereffective mobility magnitude than a nucleic acid and a higher effectivemobility magnitude than a contaminant, and (ii) a second electrolytewhich has a lower effective mobility magnitude than said contaminant. Insome embodiments of aspects provided herein, said first electrolytecomprises caproic acid. In some embodiments of aspects provided herein,said second electrolyte comprises HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In someembodiments of aspects provided herein, said kit further comprisessample buffer, wherein said sample buffer comprises leading electrolytebuffer, trailing electrolyte buffer, or urea in any combination. In someembodiments of aspects provided herein, said kit further comprises asample buffer comprising urea and thiourea.

An aspect of the present disclosure provides a method for samplepurification, comprising: (a) loading into a fluidic device (i) a tissuesample comprising nucleic acids and a contaminant, wherein said tissuesample is not an unlysed whole blood sample, (ii) a trailing electrolytebuffer comprising trailing electrolyte ions with an effective mobilityhaving a magnitude greater than a magnitude of an effective mobility ofsaid contaminant and lower than a magnitude of an effective mobility ofsaid nucleic acids, and (iii) a leading electrolyte buffer comprisingleading electrolyte ions, with a second effective mobility, wherein saidsecond effective mobility has a magnitude greater than said magnitude ofsaid effective mobility of said nucleic acids; and (b) applying anelectrical field within said fluidic device to conduct isotachophoresiswith said trailing electrolyte ions, said nucleic acids, and saidleading electrolyte ions, thereby purifying said nucleic acids from saidcontaminant in said tissue sample.

In some embodiments of aspects provided herein, said tissue sample isnot a whole blood sample. In some embodiments of aspects providedherein, said trailing electrolyte ions comprise caproic acid. In someembodiments of aspects provided herein, said leading electrolyte ionscomprise chloride. In some embodiments of aspects provided herein, saidtrailing electrolyte buffer comprises second trailing electrolyte ionshaving a different effective mobility than said first trailingelectrolyte ions. In some embodiments of aspects provided herein, saidsecond trailing electrolyte ions comprise HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In someembodiments of aspects provided herein, said second trailing electrolyteions comprise MOPS (3-(N morpholino)propanesulfonic acid) In someembodiments of aspects provided herein, said second trailing electrolyteions comprise HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)and first trailing electrolyte ions are comprised of caproic acid. Insome embodiments of aspects provided herein, said second trailingelectrolyte ions comprise MOPS (3-(N-morpholino)propanesulfonic acid)and first, trailing electrolyte ions are comprised of caproic acid. Insome embodiments of aspects provided herein, said second trailingelectrolyte ions comprise HEPES and first trailing electrolyte ionscomprise MOPS. In some embodiments of aspects provided herein, saidtrailing electrolyte buffer comprises second trailing electrolyte ionswith a second effective mobility, wherein said second effective mobilityhas a magnitude about the same as or lower than said magnitude of saideffective mobility of said contaminant. In some embodiments of aspectsprovided herein, said contaminant is selected from the group consistingof crosslinked nucleic acids, embedding material, fixation chemicals,proteins, inhibitors, and combinations thereof. In some embodiments ofaspects provided herein, said contaminant comprises crosslinked nucleicacids. In some embodiments of aspects provided herein, said tissuesample is combined with said trailing electrolyte buffer prior to saidloading. In some embodiments of aspects provided herein, said tissuesample is combined with said leading electrolyte buffer prior to saidloading. In some embodiments of aspects provided herein, said loading ofsaid leading electrolyte buffer is conducted prior to said loading ofsaid tissue sample. In some embodiments of aspects provided herein, themethod further comprises eluting an output solution comprising saidpurified nucleic acids from an outlet reservoir of said fluidic device.In some embodiments of aspects provided herein, a concentration of saidpurified nucleic acids in said output solution is at least abouttwo-fold higher than a concentration of said nucleic acids in saidtissue sample. In some embodiments of aspects provided herein, aconcentration of said crosslinked nucleic acids in said output solutionis at least about two-fold lower than a concentration of saidcrosslinked nucleic acids in said tissue sample. In some embodiments ofaspects provided herein, said output solution does not comprise saidcontaminant. In some embodiments of aspects provided herein, said tissuesample is fresh tissue. In some embodiments of aspects provided herein,said tissue sample is fresh frozen (FF) tissue. In some embodiments ofaspects provided herein, said tissue sample is formalin fixed paraffinembedded tissue (FFPE). In some embodiments of aspects provided herein,the method further comprises, prior to said loading, lysing ordisrupting said tissue sample. In some embodiments of aspects providedherein, said lysing or disrupting is conducted using urea or thiourea.

An aspect of the present disclosure provides a method for samplepurification, comprising: (a) loading into a first channel on a fluidicdevice (i) a first tissue sample comprising first nucleic acids and afirst contaminant, (ii) a first trailing electrolyte buffer comprisingfirst trailing ions, wherein a magnitude of an effective mobility ofsaid first trailing ions is less than a magnitude of an effectivemobility of said first nucleic acids, and (iii) a first leadingelectrolyte buffer comprising first leading ions, wherein a magnitude ofan effective mobility of said first leading ions is greater than saidmagnitude of said effective mobility of said first nucleic acids; (b)loading into a second channel on said fluidic device (iv) a secondtissue sample comprising second nucleic acids and a second contaminant,(v) a second trailing electrolyte buffer comprising second trailingions, wherein a magnitude of said second trailing ions is less than amagnitude of an effective mobility of said second nucleic acids, and(vi) a second leading electrolyte buffer comprising second leading ions,wherein a magnitude of an effective mobility of said second leading ionsis greater than said magnitude of said effective mobility of said secondnucleic acids; and (c) applying an electrical field within said fluidicdevice to conduct isotachophoresis in said first channel with said firsttrailing ions, said first nucleic acids, and said first leading ions,and to conduct isotachophoresis in said second channel with said secondtrailing ions, said second nucleic acids, and said second leading ions,thereby purifying said first nucleic acids from said first contaminantand purifying said second nucleic acids from said second contaminant.

In some embodiments of aspects provided herein, said first trailingelectrolyte buffer or said first leading electrolyte buffer furthercomprises a lysis agent or a tissue disruption agent. In someembodiments of aspects provided herein, said second trailing electrolytebuffer or said second leading electrolyte buffer further comprises alysis agent or a tissue disruption agent. In some embodiments of aspectsprovided herein, said lysis agent or said tissue disruption agentcomprises one or more agents selected from the group consisting of asolution with pH greater than about 12, a proteinase, urea, thiourea,and a surfactant.

An aspect of the present disclosure provides a method for samplepurification, comprising: (a) loading into a first zone of a fluidicdevice (i) a tissue sample comprising nucleic acids and a contaminant,(ii) a trailing electrolyte buffer comprising trailing ions, wherein amagnitude of an effective mobility of said trailing ions is lower than amagnitude of an effective mobility of said nucleic acids, and (iii) aleading electrolyte buffer comprising leading ions, wherein a magnitudeof an effective mobility of said leading ions is greater than saidmagnitude of said effective mobility of said nucleic acids; and (b)applying an electrical field on said fluidic device to conductisotachophoresis in a second zone of said fluidic device with saidtrailing ions, said nucleic acids, and said leading ions, therebypurifying said nucleic acids from said contaminant, wherein during saidapplying, said first zone is maintained at a first temperature and saidsecond zone is maintained at a second temperature different from saidfirst temperature.

In some embodiments of aspects provided herein, said trailingelectrolyte buffer or said leading electrolyte buffer further comprisesa lysis agent or a tissue disruption agent. In some embodiments ofaspects provided herein, said lysis agent or said tissue disruptionagent comprises one or more agents selected from the group consisting ofa solution with pH greater than about 12, a proteinase, urea, thiourea,and a surfactant. In some embodiments of aspects provided herein, saidfirst temperature is between about 4° C. and about 40° C. In someembodiments of aspects provided herein, said first temperature isbetween about 40° C. and about 80° C.

An aspect of the present disclosure provides a method for samplepurification, comprising: (a) loading into a first zone of a fluidicdevice (i) a tissue sample comprising nucleic acids, (ii) a trailingelectrolyte buffer comprising trailing ions, wherein a magnitude of aneffective mobility of said trailing ions is lower than a magnitude of aneffective mobility of said nucleic acids, and (iii) a leadingelectrolyte buffer comprising leading ions, wherein a magnitude of aneffective mobility of said leading ions is greater than said magnitudeof said effective mobility of said nucleic acids; (b) in said firstzone, conducting on said tissue sample at least one sample preparationselected from the group consisting of (1) removing embedding material,(2) disrupting tissue, (3) lysing cells, (4) de-crosslinking nucleicacids, (5) digesting proteins and (6) digesting nucleic acids; and (c)applying an electrical field within said fluidic device to conductisotachophoresis in a second zone of said fluidic device with saidtrailing ions, said nucleic acids, and said leading ions, therebypurifying said nucleic acids from a contaminant in said tissue sample.

In some embodiments of aspects provided herein, said removing embeddingmaterial or said lysing cells comprises, prior to said applying saidelectric field, incubating said tissue sample in said fluidic device ata temperature of at least about 37° C. for duration of at least about 1minute. In some embodiments of aspects provided herein, said temperatureis from about 40° C. to about 80° C. In some embodiments of aspectsprovided herein, said duration is from about 1 minute to about 60minutes. In some embodiments of aspects provided herein, said disruptingtissue or said lysing cells comprises applying mechanical stress to saidsample. In some embodiments of aspects provided herein, said disruptingtissue or said lysing cells comprises applying heat to said sample. Insome embodiments of aspects provided herein, said applying heat resultsin a temperature of said tissue sample from about 30° C. to about 65° C.In some embodiments of aspects provided herein, said disrupting tissueor said lysing cells comprises a solution pH of at least 12. In someembodiments of aspects provided herein, said disrupting tissue or saidlysing cells comprises proteolytic digestion. In some embodiments ofaspects provided herein, said proteolytic digestion is conducted at atemperature greater than about 25° C. In some embodiments of aspectsprovided herein, said temperature is from about 30° C. to about 65° C.In some embodiments of aspects provided herein, said disrupting tissueor said lysing cells comprises applying at least one surfactant to saidtissue or said cells. In some embodiments of aspects provided herein,said disrupting tissue or said lysing cells comprises applying asolution comprising urea to said tissue or said cells. In someembodiments of aspects provided herein, said solution further comprisesthiourea. In some embodiments of aspects provided herein, aconcentration of said urea in said solution is from about 4 M to about 9M and a concentration of said thiourea in said solution is from about0.5 M to about 3.5 M. In some embodiments of aspects provided herein, aconcentration of said urea in said solution is from about 6.5 M to about7.5 M and a concentration of said thiourea in said solution is fromabout 1.5 M to about 2.5 M. In some embodiments of aspects providedherein, said de-crosslinking nucleic acids comprises digestingcrosslinking proteins with proteinase K. In some embodiments of aspectsprovided herein, said digesting nucleic acids is performed with DNase orRNase.

An aspect of the present disclosure provides a method for samplepurification, comprising: (a) loading onto a fluidic device (i) a tissuesample comprising nucleic acids, wherein said tissue sample is embeddedor fixed, (ii) a trailing electrolyte buffer comprising trailingelectrolytes, wherein said trailing electrolytes have a lower effectivemobility than said nucleic acids, and (iii) a leading electrolyte buffercomprising leading electrolytes, wherein said leading electrolytes havea higher effective mobility than said nucleic acids; and (b) applying anelectrical field on said fluidic device to conduct isotachophoresis withsaid trailing electrolytes, said nucleic acids, and said leadingelectrolytes, thereby purifying said nucleic acids from a contaminant insaid tissue sample.

In some embodiments of aspects provided herein, said contaminant isselected from the group consisting of crosslinked nucleic acids,embedding material, fixation chemicals, enzymes, and inhibitors. In someembodiments of aspects provided herein, said embedding materialcomprises paraffin. In some embodiments of aspects provided herein, saidtissue sample is formalin-fixed. In some embodiments of aspects providedherein, said tissue sample is embedded and fixed. In some embodiments ofaspects provided herein, said tissue sample is a formalin-fixedparaffin-embedded (FFPE) tissue sample. In some embodiments of aspectsprovided herein, said tissue sample is a dissected tissue sample. Insome embodiments of aspects provided herein, said dissected tissuesample is dissected FFPE sample. In some embodiments of aspects providedherein, the method further comprises the step of comparing acharacteristic of said nucleic acids to nucleic acids from othersamples. In some embodiments of aspects provided herein, saidcharacteristic is an expression level. In some embodiments of aspectsprovided herein, said characteristic is a nucleic acid sequence. In someembodiments of aspects provided herein, said characteristic is amolecular weight. In some embodiments of aspects provided herein, saidcharacteristic is a nucleic acid integrity. In some embodiments ofaspects provided herein, said characteristic is a nucleic acid purity.In some embodiments of aspects provided herein, the method furthercomprises a step of administering a drug based on said characteristic ofsaid nucleic acids. In some embodiments of aspects provided herein, saidtissue sample is a tumor sample. In some embodiments of aspects providedherein, said trailing electrolyte buffer has a pH of about 7. In someembodiments of aspects provided herein, said trailing electrolyte bufferhas a pH of greater than about 7. In some embodiments of aspectsprovided herein, the method further comprises, prior to said applyingsaid electric field, incubating said tissue sample in said fluidicdevice at a temperature of at least about 37° C. for duration of atleast about 1 minute. In some embodiments of aspects provided herein,said temperature is from about 40° C. to about 80° C. In someembodiments of aspects provided herein, said duration is from about 1minute to about 60 minutes. In some embodiments of aspects providedherein, said leading electrolyte buffer comprises proteinase K. In someembodiments of aspects provided herein, the method further comprisesremoving protein crosslinks from said nucleic acids using saidproteinase K. In some embodiments of aspects provided herein, the methodfurther comprises, after said applying said electric field, removingprotein crosslinks from said nucleic acids using heat. In someembodiments of aspects provided herein, the method further compriseseluting an output solution comprising said purified nucleic acids froman outlet reservoir of said fluidic device. In some embodiments ofaspects provided herein, a concentration of said purified nucleic acidsin said output solution is at least about two-fold higher than aconcentration of said nucleic acids in said tissue sample. In someembodiments of aspects provided herein, a concentration of saidcrosslinked nucleic acids in said output solution is at least abouttwo-fold lower than a concentration of said crosslinked nucleic acids insaid tissue sample. In some embodiments of aspects provided herein, saidoutput solution does not comprise said contaminant. In some embodimentsof aspects provided herein, said output solution has a volume equal toor less than about 50 μL. In some embodiments of aspects providedherein, said tissue sample has a mass of at least about 1 ng. In someembodiments of aspects provided herein, said tissue sample has a volumeof less than about 500 μL. In some embodiments of aspects providedherein, said trailing electrolytes have a higher effective mobility thansaid contaminant. In some embodiments of aspects provided herein, saidtrailing electrolytes comprise (i) first ions, wherein said first ionshave a higher effective mobility magnitude than said contaminant, and(ii) second ions, wherein said second ions have an effective mobilitymagnitude about the same as or lower than said contaminant. In someembodiments of aspects provided herein, said conducting isotachophoresisquenches a pH of said tissue sample to about 7. In some embodiments ofaspects provided herein, the method further comprises, prior to saidloading, conducting de-paraffinization on said tissue sample. In someembodiments of aspects provided herein, said tissue sample is ahistorical formalin-fixed paraffin-embedded (FFPE) sample, furthercomprising comparing a characteristic of said nucleic acids to acharacteristic of different nucleic acids from a different tissuesample. In some embodiments of aspects provided herein, the methodfurther comprises a step of detecting a concentration of said nucleicacids. In some embodiments of aspects provided herein, saidconcentration is less than or equal to about 1 picogram per microliter(pg/μL). In some embodiments of aspects provided herein, saidconcentration is less than or equal to about 0.5 pg/μL. In someembodiments of aspects provided herein, said concentration is at leastabout 1 picogram per microliter (pg/μL).

An aspect of the present disclosure provides a fluidic device,comprising: a sample purification region, comprising: (a) a first zone;(b) a sample inlet located in said first zone; (c) a trailingelectrolyte reservoir in fluid communication with said first zone; (d) asecond zone in fluid communication with said first zone; (e) a leadingelectrolyte reservoir in fluid communication with said second zone; (f)a sample outlet in fluid communication with said second zone; (g) afirst heater in thermal communication with said first zone; and (h) asecond heater configured to transfer heat to said second zone, whereinsaid first zone is substantially thermally isolated from said secondzone.

An aspect of the present disclosure provides a fluidic device,comprising: a sample purification region, comprising: (a) a first zone;(b) a sample inlet located in said first zone; (c) a trailingelectrolyte reservoir in fluid communication with said first zone; (d) asecond zone in fluid communication with said first zone; (e) a leadingelectrolyte reservoir in fluid communication with said second zone; (f)a sample outlet in fluid communication with said second zone; and (g) aheater in thermal communication with said first zone and said secondzone.

In some embodiments of aspects provided herein, the device furthercomprises a second sample purification region. In some embodiments ofaspects provided herein, said first zone is a de-paraffinization zone.In some embodiments of aspects provided herein, said first zone is adisruption zone. In some embodiments of aspects provided herein, saidsecond zone is an isotachophoresis zone. In some embodiments of aspectsprovided herein, said first zone or said second zone has a width of lessthan about 1 mm. In some embodiments of aspects provided herein, saidfirst zone or said second zone has a width of less than about 0.5 mm.

An aspect of the present disclosure provides a kit, comprising a deviceprovided herein, a trailing electrolyte buffer comprising trailingelectrolytes, and a leading electrolyte buffer comprising leadingelectrolytes.

In some embodiments of aspects provided herein, said trailingelectrolyte buffer contains a mixture of at least two electrolytes withdifferent effective mobilities. In some embodiments of aspects providedherein, said mixture comprises (i) a first electrolyte that has a lowereffective mobility magnitude than a nucleic acid and a higher effectivemobility magnitude than a contaminant, and (ii) a second electrolytewhich has a lower effective mobility magnitude than said contaminant. Insome embodiments of aspects provided herein, said contaminant comprisescrosslinked nucleic acids. In some embodiments of aspects providedherein, said first electrolyte comprises caproic acid. In someembodiments of aspects provided herein, said second electrolytecomprises HEPES.

An aspect of the present disclosure provides a method for samplepurification, comprising: (a) loading into a fluidic device (i) a tissuesample comprising nucleic acids, (ii) a trailing electrolyte buffer,said trailing electrolyte buffer comprising trailing electrolyte ionswith a first effective mobility, wherein said first effective mobilityhas a magnitude lower than a magnitude of an effective mobility of saidnucleic acids, (iii) a first leading electrolyte buffer in a firstleading electrolyte reservoir, said first leading electrolyte buffercomprising first leading electrolyte ions with a second effectivemobility, wherein said second effective mobility has a magnitude greaterthan said magnitude of said effective mobility of said nucleic acids,and (iv) a second leading electrolyte buffer in a second leadingelectrolyte reservoir, said second leading electrolyte buffer comprisingsecond leading electrolyte ions with a third effective mobility, whereinsaid third effective mobility has a magnitude greater than saidmagnitude of said effective mobility of said nucleic acids, wherein saidfirst leading electrolyte buffer is different from said second leadingelectrolyte buffer; (b) first conducting isotachophoresis with saidtrailing electrolyte ions, said nucleic acids, and said first leadingelectrolyte ions, thereby purifying said nucleic acids from saidcontaminant in said tissue sample; and (c) second conductingisotachophoresis with said trailing electrolyte ions, said nucleicacids, and said second leading electrolyte ions.

In some embodiments of aspects provided herein, said second conductingisotachophoresis comprises changing an applied current from a firstchannel to a second channel. In some embodiments of aspects providedherein, said first leading electrolyte ions are the same as said secondleading electrolyte ions, and wherein a concentration of said firstleading electrolyte ions in said first leading electrolyte buffer isdifferent from a concentration of said second leading electrolyte ionsin said second leading electrolyte buffer. In some embodiments ofaspects provided herein, said concentration of said first leadingelectrolyte ions in said first leading electrolyte buffer is differentfrom said concentration of said second leading electrolyte ions in saidsecond leading electrolyte buffer by a factor of at least 1.5×. In someembodiments of aspects provided herein, said first leading electrolyteions are different from said second leading electrolyte ions. In someembodiments of aspects provided herein, said first leading electrolyteions are the same as said second leading electrolyte ions, and wherein aconcentration of said first leading electrolyte ions in said firstleading electrolyte buffer is the same as a concentration of said secondleading electrolyte ions in said second leading electrolyte buffer, andwherein said first leading electrolyte buffer comprises third leadingelectrolyte ions. In some embodiments of aspects provided herein, saidfirst leading electrolyte ions are the same as said second leadingelectrolyte ions, and wherein a concentration of said first leadingelectrolyte ions in said first leading electrolyte buffer is the same asa concentration of said second leading electrolyte ions in said secondleading electrolyte buffer, and wherein said second leading electrolytebuffer comprises third leading electrolyte ions. In some embodiments ofaspects provided herein, the method further comprises collecting saidnucleic acids in said second leading electrolyte reservoir. In someembodiments of aspects provided herein, the method further comprisesremoving said nucleic acids from said second leading electrolytereservoir. In some embodiments of aspects provided herein, said trailingelectrolyte buffer is loaded into a trailing electrolyte reservoir thatis separate from said first leading electrolyte reservoir and saidsecond leading electrolyte reservoir. In some embodiments of aspectsprovided herein, said first conducting isotachophoresis and said secondconducting isotachophoresis are performed by applying one electricfield. In some embodiments of aspects provided herein, said firstconducting isotachophoresis and said second conducting isotachophoresisare performed by applying more than one electric field.

An aspect of the present disclosure provides a fluidic device,comprising: a sample purification region, comprising: (a) a channelcomprising a first zone and a second zone in fluid communication withsaid first zone; (b) a sample inlet, a trailing electrolyte reservoircomprising a trailing electrolyte buffer, and a first leadingelectrolyte reservoir comprising a first leading electrolyte buffer,each in fluid communication with said first zone; and (c) a secondleading electrolyte reservoir comprising a second leading electrolytebuffer, wherein said second leading electrolyte buffer is in fluidcommunication with said second zone and wherein said second leadingelectrolyte buffer is different from said first leading electrolytebuffer.

In some embodiments of aspects provided herein, said sample inlet iscapable of receiving a sample comprising at least some non-liquidbiological material. In some embodiments of aspects provided herein,said second leading electrolyte buffer comprises a different leadingelectrolyte co-ion than said first leading electrolyte buffer. In someembodiments of aspects provided herein, said first leading electrolytebuffer comprises first leading electrolyte ions and said second leadingelectrolyte buffer comprises second leading electrolyte ions that arethe same as said first leading electrolyte ions, and wherein aconcentration of said first leading electrolyte ions in said firstleading electrolyte buffer is different from a concentration of saidsecond leading electrolyte ions in said second leading electrolytebuffer. In some embodiments of aspects provided herein, said firstleading electrolyte buffer comprises first leading electrolyte ions andsaid second leading electrolyte buffer comprises second leadingelectrolyte ions, and wherein said concentration of said first leadingelectrolyte ions in said first leading electrolyte buffer is differentfrom said concentration of said second leading electrolyte ions in saidsecond leading electrolyte buffer by a factor of at least 1.5×. In someembodiments of aspects provided herein, said first leading electrolytebuffer comprises first leading electrolyte ions and said second leadingelectrolyte buffer comprises second leading electrolyte ions that aredifferent from said first leading electrolyte ions. In some embodimentsof aspects provided herein, said first leading electrolyte buffercomprises first leading electrolyte ions and said second leadingelectrolyte buffer comprises second leading electrolyte ions that arethat same as said first leading electrolyte ions, and wherein aconcentration of said first leading electrolyte ions in said firstleading electrolyte buffer is the same as a concentration of said secondleading electrolyte ions in said second leading electrolyte buffer, andwherein said first leading electrolyte buffer comprises third leadingelectrolyte ions. In some embodiments of aspects provided herein, saidfirst leading electrolyte buffer comprises first leading electrolyteions and said second leading electrolyte buffer comprises second leadingelectrolyte ions that are the same as said first leading electrolyteions, and wherein a concentration of said first leading electrolyte ionsin said first leading electrolyte buffer is the same as a concentrationof said second leading electrolyte ions in said second leadingelectrolyte buffer, and wherein said second leading electrolyte buffercomprises third leading electrolyte ions.

An aspect of the present disclosure provides a method, comprising: (a)providing an electrokinetic fluidic device comprising a reservoir influidic communication with a channel; (b) loading a sample volume intosaid reservoir; (c) moving at least 50% of said sample volume from saidreservoir to said channel; and (d) applying an ionic current throughsaid channel.

In some embodiments of aspects provided herein, said moving is conductedwith the aid of gravity. In some embodiments of aspects provided herein,said ionic current does not substantially pass through said reservoir.In some embodiments of aspects provided herein, said at least 50% ofsaid sample volume comprises at least 80% of said sample volume. In someembodiments of aspects provided herein, said sample volume comprisesnucleic acids. In some embodiments of aspects provided herein, saidsample volume comprises a tissue sample. In some embodiments of aspectsprovided herein, said sample volume comprises a formalin-fixedparaffin-embedded (FFPE) sample. In some embodiments of aspects providedherein, said applying an ionic current comprises conductingisotachophoresis (ITP).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entiretiesto the same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A shows an exemplary protocol for sample processing and nucleicacid extraction or purification.

FIG. 1B shows an exemplary protocol for automated sample processing andnucleic acid extraction or purification.

FIG. 2A shows an exemplary schematic of isotachophoretic separation andpurification of DNA and RNA from contaminants.

FIG. 2B shows an exemplary schematic of isotachophoretic separation andpurification of nucleic acids from paraffin and other possible samplecontaminants concurrent with proteinase-mediated tissue disruption anddecrosslinking of nucleic acids.

FIG. 3 shows exemplary results of DNA extraction and purification byautomated isotachophoresis in a fluidic device compared to exemplaryresults from a typical solid phase column extraction kit.

FIG. 4A shows exemplary results for unbiased (e.g., with respect tosequence) extractions of GC-rich and AT-rich synthetic DNAoligonucleotides mixed at sample concentration ratios usingisotachophoresis.

FIG. 4B shows exemplary results for unbiased (e.g., with respect to sizeor molecular weight), pre- and post-purification, of a DNA molecularweight ladder using isotachophoresis; comparisons to two solid phasecolumn based nucleic acid purification methods are shown.

FIG. 5A shows an exemplary schematic of a channel with a samplepreparation zone and an isotachophoretic purification zone.

FIG. 5B shows an exemplary fluidic device cartridge comprising eightparallel fluidic channels and reservoirs for simultaneous processing ofup to eight samples as shown in FIG. 5A.

FIG. 5C shows an exemplary top view schematic of one channel and itsconnected reservoirs for a fluidic device cartridge as shown in FIG. 5B,further exemplifying use of gas ports for external pressure or vacuumapplication to the channels within the fluidic device cartridge.

FIG. 5D shows an exemplary side view schematic of a fluidic devicecartridge as shown in FIG. 5B.

FIG. 5E shows an exemplary end view schematic of a fluidic devicecartridge as shown in FIG. 5B.

FIG. 6A shows an exemplary top view schematic of a fluidic devicecartridge.

FIG. 6B shows an exemplary side view schematic of a fluidic devicecartridge.

FIG. 6C shows an exemplary bottom view schematic of a fluidic devicecartridge.

FIG. 6D shows an exemplary top full view schematic in three dimensionsof a fluidic device cartridge.

FIG. 7A shows an exemplary top view schematic of a fluidic devicecartridge.

FIG. 7B shows an exemplary side view schematic of a fluidic devicecartridge.

FIG. 7C shows an exemplary bottom view schematic of a fluidic devicecartridge.

FIG. 7D shows an exemplary bottom full view schematic in threedimensions of a fluidic device cartridge.

FIG. 8A shows an exemplary top view schematic of a fluidic devicecartridge.

FIG. 8B shows an exemplary side view schematic of a fluidic devicecartridge.

FIG. 8C shows an exemplary bottom view schematic of a fluidic devicecartridge.

FIG. 8D shows an exemplary bottom full view schematic in threedimensions of a fluidic device cartridge.

FIG. 9A shows an exemplary schematic of a fluidic device cartridgecomprising eight parallel channels as shown in FIG. 5B.

FIG. 9B shows an exemplary schematic of two thermal controllers, eachaligned with a zone of the eight parallel channels shown in FIG. 9A.

FIG. 10A shows an exemplary gas channel which may comprise a capillarybarrier.

FIG. 10B is a magnified schematic of the gas channel of FIG. 10A.

FIG. 11 shows an exemplary low-loss sample reservoir.

FIG. 12A shows an exemplary mechanical member which can be used to applypressure to close the channels of a fluidic device.

FIG. 12B shows an exemplary comb-like mechanical member.

FIG. 12C shows the alignment of a comb-like mechanical member and thechannels of a fluidic device.

FIG. 13A shows an exemplary benchtop device for conducting automatedsample preparation and isotachophoresis on a fluidic device cartridge.

FIG. 13B shows an exemplary computer control system that is programmedor otherwise configured to implement methods provided herein.

FIG. 14 shows exemplary results of fluorescence-based measurements andquantitation for a titration series of nucleic acids usingisotachophoresis.

FIG. 15 shows a schematic of an exemplary design of a fluidic channelwith connected reservoirs, contactless electrode(s) (may be used asconductivity sensor) and gas port(s) for conducting automated fluidloading into channel/device and automated isotachophoresis.

FIG. 16 shows a graph of voltage measurement over time in an ITP channelduring a run.

FIG. 17 shows two graphs of derivative analysis of the voltagemeasurements from FIG. 16.

FIG. 18 shows an example of conductivity measurement over time in an ITPchannel near an elution reservoir.

FIG. 19 shows an exemplary schematic of a C4D sensor implementation.

FIG. 20A shows an exemplary temperature map of an ITP channel takenusing a thermal imaging camera.

FIG. 20B shows a plot of temperature over time at the position of Cursor1 in FIG. 20A.

FIG. 21 shows a graph of temperature measurement and temperaturederivative over time during an ITP run.

FIG. 22A shows an exemplary schematic of a vertical (or column) ITPsetup.

FIG. 22B shows an exemplary image of a vertical ITP set up with an DNAITP band.

FIG. 23 shows exemplary images and corresponding fluorescence intensitytraces of extraction and separation of amplifiable (e.g., decrosslinked)DNA from crosslinked DNA from an FFPE sample using isotachophoresis.

FIG. 24A shows an exemplary image of DNA extraction and purificationfrom FFPE samples using isotachophoresis.

FIG. 24B shows exemplary DNA yields measured by quantitative PCR forextraction and purification of DNA from FFPE samples usingisotachophoresis compared to exemplary results from a typical solidphase column extraction kit.

FIG. 25A shows an image of a single channel ITP chip loaded with nucleicacid (RNA extraction and digest from human cells) stained with dye forvisualization.

FIG. 25B shows an image of a single channel ITP chip loaded with nucleicacid (RNA extraction and digest from human cells) stained with dye forvisualization.

FIG. 26A shows an image of an RNA ITP band in a chip channel duringpurification.

FIG. 26B shows an image of a total nucleic acid ITP band in a chipchannel during purification.

FIG. 26C shows a graph of an RNA quality electropherogram for the sampleshown in FIG. 26A.

FIG. 26D shows a graph of an RNA quality electropherogram for the sampleshown in FIG. 26B.

FIG. 27A shows results of DNA yield (ng) for ITP (square) compared tocolumn (diamond, Qiagen QiaAmp) extraction of whole mouse blood as afunction of percent by volume of whole blood in starting sample.

FIG. 27B shows an image of total nucleic acid in an ITP band during ITPpurification of lysed whole mouse blood on a chip.

FIG. 27C and FIG. 27D show white light and fluorescence overlay imagesof ITP chip channels showing physical separation of heme in thesample/leading electrolyte channel from the elution channel andreservoir, before and after ITP purification of 50% by volume wholeblood lysate. Nucleic acid is stained with green dye for visualizationin elution well. FIG. 27C shows the chip before ITP (blood lysate andITP buffers loaded in chip; buffer only in elution well). FIG. 27D showsthe chip after ITP (blood lysate and ITP buffers loaded in chip;purified DNA in elution well).

FIG. 27E shows an chip post ITP purification (50% by volume blood).

FIG. 27F shows an chip post ITP purification (25% by volume blood).

FIG. 28 shows results of high molecular weight DNA purification for ITPcompared to solid phase extraction.

FIG. 29A shows a fluidic device comprising 8 closed channels.

FIG. 29B shows a zoomed in microscopic view the second channel closurelocation adjacent the elution reservoir of each of the channels.

FIG. 29C shows the percent closure calculated as a function of forceapplied to the fluidic device.

FIG. 29D shows the results of conductivity measurements of channelclosure.

FIG. 30 a graph of voltage measurement and voltage derivative over timeduring an ITP run.

FIG. 31A shows a micrograph of ITP bands with focused DNA in each of 8samples in the sample channel region of the device.

FIG. 31B shows independent voltage signal data at fixed currents foreach of the 8 channels over time.

FIG. 31C shows a micrograph of the same 8 ITP bands with focused DNAfrom the samples eluted in the elution reservoir.

FIG. 31D is a magnified section of the voltage tracing (monitoring) usedfor triggering shown in FIG. 31B.

FIG. 32A shows an exemplary method for sample preparation from culturedmammalian cells and ITP for DNA purification.

FIG. 32B shows an exemplary method for sample preparation from tissuesamples and ITP for DNA purification.

FIG. 33 shows an exemplary process workflow for automated ITP.

FIG. 34 shows a circuit configured to detect and prevent current leakageduring ITP.

FIGS. 35A-35B show an exemplary fluidic device which comprises twointerlocking parts.

FIGS. 36A-36B show an example of a fluidic device which comprisesmultiple parts.

FIGS. 37A-37B show an example of a fluidic device which comprises threeparts.

FIG. 38 shows an exemplary channel schematic for 8 parallel channels onthe underside of the chip of a three part fluidic device.

FIG. 39 shows an exemplary multi-part fluidic device.

FIG. 40 shows an exemplary fluidic circuit comprising voltage andtemperature sensing.

FIGS. 41A-41B show an exemplary “cliff capillary barrier”.

FIGS. 42A-42B show an exemplary “plateau capillary barrier”.

FIG. 43 shows an exemplary channel or fluidic circuit highlighting theinitial fluid interface positions after loading.

FIG. 44 shows an exemplary channel or fluidic circuit highlighting thefinal fluid interface positions after loading.

FIG. 45A shows an example of the fluidic layer of the chip.

FIG. 45B shows a cutaway of the pneumatic port.

FIG. 45C shows an implementation of this design.

FIG. 46A shows an exemplary microfluidic channel in which an emptysample channel can be detected.

FIG. 46B shows the sequence of events for detection of whether thischannel is empty or filled.

FIG. 46C shows pressure traces and control signal traces from theimplementation of this technique.

FIGS. 47A-47B show an exemplary pneumatic control scheme for stagedliquid loading.

FIGS. 48A-48H show exemplary sample inlet reservoir designed for directinjection.

FIGS. 49A and 49B show an exemplary low-dispersion elution channel.

FIG. 50A1 shows a fluidic surface of a substrate with channelterminating in through-holes that communicate with a reservoir on anopposing, reservoir surface of the substrate.

FIGS. 50A-50B show technical drawings of an exemplary elution reservoiron the chip device.

FIG. 50C-50E are background subtracted fluorescence images of thefluidic device at different steps of elution for design in FIG. 50A.

FIG. 50F is a block diagram that represents the steps in the elutionworkflow.

FIG. 51A shows an exemplary fluidic reservoir which can be used fornucleic acid elution.

FIG. 51B shows a second embodiment of the reservoir, with furtherchanges for injection molding compatibility.

FIGS. 51C and 51D show two views of a third embodiment of this reservoirdesign.

FIG. 51E shows a comparison of the residence time of nucleic acidbetween the disclosed design and a reference design.

FIG. 51F shows a cross-section of the reference design. It is a draftedvertical cylinder without any internal structure.

FIGS. 52A-52D show a fluidic reservoir consisting of an ellipticalthrough-hole, dimensioned such that a pipette tip can be easilypositioned for reliable fluid recovery from said device.

FIGS. 53A-53C show another exemplary fluidic reservoir.

FIG. 53D shows a top view of an exemplary elution reservoir. FIG. 53Eshows a section view of the elution reservoir of FIG. 53D.

FIG. 54A shows an exemplary mechanical member which can be used to applypressure to close the channels of a fluidic device.

FIG. 54B shows an exemplary ridge-like mechanical member.

FIG. 54C shows the alignment of a ridge-like mechanical member and thechannels of a fluidic device.

FIG. 54D shows a break out component assembly diagram for the mechanicalactuator coupled to a ridge-like structure for closing channels.

FIG. 55 depicts a pneumatic control block diagram for beta prototype andproduction instrument.

FIGS. 56A-56B and 58A-58C show alternative mechanisms for closingchannels without the use of heat or pressure-induced plastic deformationof a fluidic device.

FIGS. 59A-59C show an exemplary benchtop device for conducting automatedisotachophoresis and/or sample preparation on a fluidic devicecartridge.

FIG. 60 shows an exemplary image which may be displayed to a user toinstruct and guide the user through the reservoir loading process.

FIG. 61 shows a pneumatic manifold which may facilitate engagement ofthe microfluidic chip with the instrument.

FIG. 62 shows a cross-section of a chip in an instrument.

FIG. 63 depicts a vertical manifold motion mechanism comprising amechanical assembly design for motion with alignment andauto-retraction.

FIG. 64 shows a design for a horizontal manifold motion mechanismincluding a mechanical assembly design for horizontal motion using arack and pinion.

FIG. 65 depicts a heat pipe with thermoelectric cooler design forkeeping an area at a prescribed temperature remote from the location ofthe thermoelectric cooler.

FIG. 66 shows another exemplary benchtop device for conducting automatedisotachophoresis and/or sample preparation on a fluidic devicecartridge.

FIG. 67 shows a plot of peak area response to nucleic acid mass forvarious nucleic acid binding dyes.

FIG. 68 shows a plot of peak width response to nucleic acid mass forvarious nucleic acid binding dyes.

FIGS. 69A-69C depict Quantifluor® incompatibility with PCR.

FIG. 70 depicts PicoGreen compatibility with Qubit dsDNA Assay.

FIG. 71 depicts Syto13 compatibility with Qubit dsDNA Assay.

FIGS. 72A-72B depict PicoGreen compatibility with PCR.

FIGS. 73A-73C depicts Syto13 compatibility with PCR. gDNA was purifiedusing isotachophoresis in the presence and absence of Syto13.

FIG. 74 depicts PicoGreen compatibility with amplicon-based sequencinglibrary prep.

FIG. 75 shows Syto13 compatibility with amplicon-based sequencinglibrary prep.

FIGS. 76A-76B depict PicoGreen incompatibility with whole genomesequencing library prep.

FIGS. 77A-77B depict Syto13 compatibility with next generationsequencing library prep.

FIG. 78 shows PicoGreen incompatibility with whole genome sequencinglibrary prep.

FIG. 79 depicts Syto13 compatibility with next generation sequencinglibrary prep.

FIGS. 80A-80D depict Syto13 compatibility with whole exome nextgeneration sequencing library prep.

FIG. 81 shows a block diagram that describes an optical signalprocessing algorithm.

FIG. 82 shows a drawing of a mechanical optical assembly design forillumination and detection of the florescence of a sample bound to adye.

FIG. 83 shows the optical path that achieves excitation of a samplebound dye fluorescence and the capture of the emitted light from thesample bound dye fluorescence.

FIGS. 84A-84F show a control scheme for how an electrical circuitcreated by electrodes in a channel may be verified.

FIG. 85 shows a channel closer—tooth like member with mechanicalactuator

FIG. 86 shows contrast images of dyed analyte material in the channel.

FIG. 87A shows conductivity data that was obtained by using aconductivity meter to measure the conductivity of eluted material.

FIG. 87B shows contrast images of dyed analyte material in the channel.

FIG. 88A shows a typical trace of temperature signal captured by IRsensor during the run.

FIG. 88B shows the first-order derivative values of the temperature datain Figure A.

FIG. 88C shows the residual of the data minus the fit (0016) and thedata minus the null hypothesis (0015) are compared to produce alikelihood ratio.

FIG. 88D shows a block diagram of triggering process.

FIG. 88E shows a successful triggering of a nucleic acid extractionprocess.

FIG. 88F shows a failed triggering run.

FIG. 89A shows the capillary barrier between sample (in this case,sample prepared in leading electrolyte) and leading electrolyte buffer.

FIG. 89B shows the passage of a nucleic acid with adding a slow ion(3-(N-morpholino)propanesulfonic acid) and a control.

FIG. 89C demonstrates the nucleic acid morphology sometime later in theextraction process.

FIG. 89D shows the capillary barrier between leading electrolyte bufferand elution buffer.

FIG. 89E shows examples of adjusting buffer interfaces.

FIG. 89F shows the facilitation of nucleic acid passage by adjustinginterfaces position.

FIGS. 90A-90C show the lysis efficiency of three different cells lines.

FIGS. 91A-91B show exemplary temperature measurement results using aninfra-red thermal sensor to trigger a reduction or elimination of anelectric current in one of the channels.

FIG. 92A shows a block diagram of sample channel to LE channeltriggering process used.

FIG. 92B shows exemplary traces of the voltage, the derivative of thevoltage, and the measurement error used for sample channel to LE channeltriggering.

FIG. 92C shows a block diagram of LE channel triggering process used.

FIG. 92D shows exemplary traces of the voltage, the derivative of thevoltage, and the measurement error used for triggering at the narrowingafter the capillary barrier within the LE channel.

FIG. 92E shows a block diagram of the elution triggering process used.

FIG. 92F shows exemplary traces of the voltage, the derivative of thevoltage, and the measurement error used for elution triggering.

DETAILED DESCRIPTION Overview

Sample preparation is a first step to almost all genomic andtranscriptomic analyses, and yet can be a primary source of analysisvariability. Sample preparation can also be manually intensive,particularly when the sample is a formalin-fixed paraffin-embedded(FFPE) sample containing cross-linked proteins.

The present disclosure provides processes and devices to improve theefficiency of nucleic acid extraction and purification from tissue andcellular samples, including samples that have been processed in someway, such as paraffin-embedded samples or chemically-fixed samples(e.g., FFPE samples, samples that contain solid tissue). Methodsprovided herein include methods of on-chip or off-chip preparation ofsuch processed samples prior to conducting isotachophoresis usingmethods that incorporate leading electrolyte ions and trailingelectrolyte ions. In some instances, the methods include treating (e.g.,by removal of embedding material, lysis, enzymatic disruption) a fixedsolid tissue in a trailing electrolyte buffer or leading electrolytebuffer prior to conducting isotachophoresis on the sample. The methodscan also include use of a second leading electrolyte buffer of lowerionic strength in order to produce a sample compatible with downstreamprocesses like amplification or other enzymatic assays. The devices andsystems provided herein include devices suitable for conductingisotachophoresis on samples derived from tissues, including microfluidicdevices with parallel processing features and automated feedback-controlmechanisms that may include thermal sensors that detect changes intemperature within sample processing channels.

The processes and devices of the present disclosure can provide improvednucleic acid recovery from a sample, especially from low abundancesamples (e.g., less than 100 ng of nucleic acid), samples withrelatively high volumes (e.g., total volume greater than 25 μl, totalvolume greater than 50 μl, total volume greater than 100 μl, or more) orliquid samples containing solid particles. The processes and devicesprovided herein also can provide high repeatability, and reduced biasfor short nucleic acids. The devices provided herein can integratesample preparation (e.g., removal of crosslinking or embedding material)and nucleic acid extraction operations within one device. Devices andprocesses of the present disclosure can also provide for compatibilitywith process automation, integration with downstream processes,integration with in-line quantitation (e.g., at single picogramresolution), and/or integration with nucleic acid length and sequencedistribution analysis.

The methods provided herein are often methods of performingisotachophoresis under conditions suitable to extract nucleic acids fromcertain samples, especially FFPE samples. In some instances, thedisclosed methods include methods of performing isotachophoresis using atrailing electrolyte buffer containing at least two ions with differentmagnitudes of effective mobilities. The methods may also include methodsof conducting isotachophoresis using two different leading electrolytebuffers, one of which may serve as a sample elution buffer. The methodscan include process automation and parallel processing of multiplesamples.

The present disclosure also includes protocols using buffer and spacerchemistries. These buffer and spacer chemistries can include the use ofmultiple species of electrolytes for conducting ITP. For example, thetrailing electrolytes can comprise a mixture of electrolyte species,capable of separating non-crosslinked nucleic acids from crosslinkednucleic acids, while separating either non-crosslinked nucleic acids orboth crosslinked and non-crosslinked nucleic acids from contaminantswithin a sample.

The devices provided herein include injection-molded fluidic deviceswith parallel sample processing channels capable of performing ITP in amultiplexed fashion and ITP devices with two or more regions that areconnected to a thermal device. Techniques of the present disclosure canemploy ITP to simultaneously collect, purify, and focus extracted RNAand DNA, to quantify total extracted nucleic acid on-chip (e.g., viain-line ITP-aided concentration into very small volumes or labeling withan intercalating fluorescent dye), and to deliver nucleic acidsdownstream to parallel output reservoirs compatible with roboticpipetting.

Techniques of the present disclosure can enable purification of samplematerial (e.g., nucleic acids) without binding the sample material to asolid support. Techniques of the present disclosure can enablepurification of sample material (e.g., nucleic acids) without the use ofliquid-phase extraction. This can enable purification without dependenceon solubility differences.

The operation of devices of the present disclosure can be automated,largely automated, or partly automated. In some cases, methods of thepresent disclosure involve only a single off-chip mixing step ofdispensing a sample (e.g., FFPE section) into a solution (e.g., alkalinesolution, lysis solution, or buffered solution comprising urea and/orthiourea), followed by loading of the sample into a reservoir of afluidic device for further on-device sample preparation (e.g.deparaffinization, tissue disruption and cell lysing, proteasedigestion, proteolytic digestion, or other treatment including proteindenaturation, or nuclease digestion) and nucleic acid extraction,purification, enrichment, in-line quantitation, and sizing orfractionation (e.g., size selection). In some cases, methods of thepresent disclosure include dispensing a sample (e.g., FFPE section orother tissue sample) into a reservoir or channel of a fluidic device(e.g., cartridge) pre-filled with a solution (e.g., alkaline solution,lysis solution, or buffered solution comprising urea and/or thiourea)for on-device sample preparation (e.g. deparaffinization, tissuedisruption and cell lysing, protease digestion or other treatmentincluding protein denaturation, or nuclease digestion) and nucleic acidextraction, purification, enrichment, in-line quantitation, and sizingor fractionation (e.g., size selection). In some cases, methods of thepresent disclosure include disruption tissue and/or lysing cells of asample off-chip, followed by loading of the sample, which may behomogenous or a non-homogenous mixture of lysed solid tissue and nucleicacids, into a reservoir of a fluidic device for further on device samplepreparation (e.g. deparaffinization, protease digestion or othertreatment including protein denaturation, or nuclease digestion) andnucleic acid extraction, purification, enrichment, in-line quantitation,and sizing or fractionation (e.g., size selection). Nuclease digestioncan include removal of DNA for DNA-free RNA extractions or removal ofRNA for RNA-free DNA extractions. The fluidic devices provided hereincan be used with a benchtop system to automate an electric-field-basedmethod for the extraction of DNA and RNA from samples.

Devices of the present disclosure include systems that can automate andintegrate on-chip heating (e.g., to a temperature from 37° C. to 80°C.), sample preparation (e.g., deparaffinization, tissue disruption andcell lysing), buffer exchange, nucleic acid extraction and purification,enrichment of uncrosslinked or amplifiable nucleic acids (e.g., byseparating it away and delivering it separately from crosslinked nucleicacids), and delivery of purified nucleic acids to an output reservoir,such as an array compatible with manual or robotic pipetting. Forexample, the present disclosure includes an eight-channel cartridge in astandard, robotic automation compatible microtiter plate format, as wellas integrated benchtop controller prototypes that can afford automatedcontrol of loading of buffers and other fluids, application oftemperature and electric fields to the device, and automated start andend run processing of samples in parallel. This system can be easilymodified in the future, as needed, to afford higher throughput for usein larger, diagnostic or clinical labs (e.g., 96-well sample format).

For example, FIG. 1A shows an exemplary process diagram for sampleprocessing and nucleic acid extraction using techniques of the presentdisclosure. A sample can be provided 101 and subjected to anypre-processing steps 102, such as mixing with a buffer, lysis, orremoval of embedding material (if present). The sample (and, forexample, buffer) can then be loaded onto a fluidic device 103. Samplepreparation steps 104 can then be performed on the fluidic device, suchas removal of embedding material (if present and if not previouslyremoved during pre-processing), tissue disruption, cell lysis, proteinor proteolytic digestion and (for example) nuclease digestion.Isotachophoresis 105 can then be performed to separate and purifynucleic acids from contaminants within the sample (e.g. cell debris,plasma membranes, small molecules, embedding material, crosslinkednucleic acids, fixatives such as formalin, inhibitors, enzymes such asdigestion or restriction enzymes). Other steps can occur concurrentlywith isotachophoresis, such as de-crosslinking of crosslinked nucleicacids (e.g. with heat or protease digestion). Nucleic acids can bedetected and quantified 106 during or subsequent to isotachophoresis.Once extracted or purified, nucleic acids can then be eluted andrecovered from the device 107.

FIG. 32A shows a non-limiting exemplary method for sample preparationfrom cultured mammalian cells and ITP for DNA purification utilizing themethods and devices provided herein. In general, the steps may includeisolation (Step 151) and washing of cells (Step 152), cell lysis (Steps153-155) and protein degradation (Steps 156-157), and homogenization ofliberated DNA in a lysate (Steps 158-159), all while maintainingappropriate ionic content for downstream ITP.

At Step 151, cultured mammalian cells may be pelleted from live, healthy(e.g. >90% viability), log-phase cell culture by centrifugation (e.g.250 g×5 min). Pelleting may be preceded by trypsinization in at leastsome instances, for example when using adherent or semi-adherent cells.The spent media may be discarded before washing the cells in freshmedia, pelleting the cells, resuspending the cells in fresh media, andcounting the cells. A cell suspension of appropriate density (e.g.100,000 live cells per lane of ITP extraction) may be deposited into amicrocentrifuge tube (e.g. a 2 ml Eppendorf Lo-Bind microcentrifugetube) for downstream processing (e.g. Steps 152-163 below). In someinstances, as will be apparent to one of ordinary skill in the art,fluorescence-activated cell sorting (FACS) of the re-suspended may beused to isolate and count cells of interest into a recipient tube whichmay then be prepared as described herein for ITP.

At Step 152, the cells may be washed with a buffer such as phosphatebuffered-saline (PBS). The cells may be pelleted by centrifugation (e.g.at 250 g×5 min). The cell culture media may then be discarded and thepellet may be resuspended in PBS (e.g. 190 uL 1×PBS (no Ca2+ or Mg2+)).The resuspended cells may be centrifuged to form a pellet and the PBSsupernatant may be removed from the pelleted cells.

At Step 153, the cell pellet may be lysed through pipet resuspension(e.g. by pipetting 5 times using a P1000 pipette) in a lysis buffer, forexample in a proprietary alkaline CCD Lysis buffer (“L1”). Lysis mayalternatively or in combination be performed using other lysistechniques which will be known to one of ordinary skill in the art, forexample via sonication, manual grinding, beadbeating, homogenization,freezing, enzymatic digestion, and/or chemical disruption. The lysedcells may then be vortexed to mix (e.g. for 3 sec). Generally, at step153, the lysis buffer is highly alkaline. In some cases, an alkalinesolution may comprise 30-120 mM NaOH (in some cases, 40-80 mM NaOH) at apH of about 10-13. An exemplary alkaline solution may comprise 80 mMNaOH, 11 mM DTT, and 0.5% v/v Igepal CA-630.

At Step 154, the cells may be incubated in the lysis buffer at roomtemperature (e.g. for 2 min) in order to allow the lysis process to lysethe cells.

At Step 155, lysis may be stopped and the sample pH may be neutralized.For example, a proprietary acidic Quench buffer (“Quench”) may beapplied to the lysed cell sample. In some cases, the Quench buffer maycontain LE only, both LE and TE, or TE only. In some cases, includingboth LE and TE or TE only in the Quench buffer may reduce retention ofnucleic acids at a capillary barrier, particularly a capillary barrierbetween the sample and LE. In some cases, quenching as described in step155 is not performed in a method disclosed herein (e.g., for highmolecular weight DNA applications). For example, it may not be necessarywhen a lysis solution is within a neutral or non-alkaline pH. In suchcases, the LE, LE and TE, or TE may be included in the lysis solution.Quenching is generally useful when an alkaline lysis solution is usedduring the lysis step (e.g., for cultured cell applications or primaryhuman cells).

The sample may be mixed by pipetting (e.g. by pipetting up and down fivetimes) and then vortexed (e.g. for 3 sec).

At Step 156, the lysed cells suspension may be treated to denatureproteins in the cell lysate. Optionally, RNA (or DNA if the samplemolecule of interest for ITP is RNA) may be degraded. For example,Proteinase K (e.g. 20 mg/mL) and optionally RNAse A (e.g. 10 mg/mL) maybe added. The sample may be vortexed (e.g. for 3 sec) to mix and thenpulse-spinned before incubation at room temperature (e.g. for 2 min).

At Step 157, the sample may be incubated at 56° C. incubation for 10min.

At Step 158, the sample may be homogenized by vortexing (e.g. for 3 sec)and pulse-spinning.

At Step 159, the sample may then be cooled to room temperature (e.g. for5 min). In some cases, where samples are prepared in LE, a TE can beadded after cooling to reduce or minimize retention of nucleic acids ata capillary barrier, particularly a capillary barrier between a sampleand LE.

At Step 160, a sample surfactant (e.g. MOPS) may optionally be added tothe sample.

At Step 161, the sample may optionally be frozen for use at a laterdate.

At Step 162, the sample may be warmed to 20° C.

At Step 163, a nucleic acid dye or stain may optionally be added to thesample lysate for visualization and detection (e.g., for quantitation ofnucleic acid mass) of nucleic acid. For example, the addition of anucleic acid stain, such as a nucleic acid binding or intercalating dye,may be used when in-line quantitation of nucleic acid mass is performedduring the ITP process.

At Step 164, the sample may be loaded into one or more samplereservoirs. Prior to loading, the sample may be pulse vortexed (e.g.twice) and pulse-spinned to agitate and mix the sample. The ITP channelmay be pre-primed with leading electrolyte buffer, trailing electrolytebuffer, and/or other buffers as described herein. Alternatively, thesample may be loaded at the same time as the rest of the liquids in thechannel.

At Step 165, ITP may be performed. During the ITP process, the DNAwithin the sample may be purified (i.e. concentrated) as it movesthrough the channel until it reaches the elution reservoir as describedherein. The sample DNA may be quantified as described herein.

At Step 166, the eluted sample may be recovered by elution (e.g. bypipetting) from the elution reservoir.

FIG. 32B shows a non-limiting exemplary method for sample preparationfrom tissue samples (e.g. fresh or FFPE tissue samples) and ITP for DNApurification utilizing the methods and devices provided herein. Ingeneral, the steps may include trimming excess paraffin from the tissue(e.g. when using slide-mounted FFPE tissue section)(Step 171), tissuelysis (Steps 172-175) and protein degradation (Steps 176-177), andreversing crosslinking of liberated DNA in the lysate (Steps 178-179),all while maintaining appropriate ionic content for downstream ITP.

At Step 171, excess paraffin may optionally be trimmed or removed fromthe FFPE tissue sample (e.g., prior to further sample preparationsteps). An FFPE tissue sample may be acquired directly from a paraffinblock or from a slide-mounted section.

At Step 172, the tissue may be collected in a microcentrifuge tube (e.g.an Eppendorf Lo-Bind microcentrifuge tube). Collection may optionallycomprise scraping the FFPE tissue sample off of a slide or otherwiseplacing a fresh or FFPE tissue sample in the tube.

At Step 173, the tissue may be lysed through pipet resuspension (e.g. bypipetting 5 times using a P1000 pipette) in a lysis buffer, for examplein a proprietary alkaline CCD Lysis buffer (“L1”). Lysis mayalternatively or in combination be performed using other lysistechniques which will be known to one of ordinary skill in the art, forexample via sonication, manual grinding, beadbeating, homogenization,freezing, enzymatic digestion, and/or chemical disruption. The lysedtissue may then be vortexed to mix (e.g. for 5 sec). Generally, at step173, the lysis buffer is highly alkaline. In some cases, an alkalinesolution may comprise 30-120 mM NaOH (in some cases, 40-80 mM NaOH) at apH of about 10-13. An exemplary alkaline solution may comprise 80 mMNaOH, 11 mM DTT, and 0.5% v/v Igepal CA-630.

At Step 174, the tissue may be incubated in the lysis buffer at 80° C.(e.g. for 3 min) in order to allow the lysis process to lyse the tissue.The sample may then be mixed by pipetting (e.g. by pipetting up and downfive times) and then vortexed (e.g. for 3 sec).

At Step 175, the lysed tissue may be incubated at room temperature (e.g.for 3 min) to cool.

At Step 176, the lysed tissue suspension may be treated to denatureproteins in the tissue lysate. Optionally, RNA (or DNA if the samplemolecule of interest for ITP is DNA) may be degraded. For example,Proteinase K (e.g. 20 mg/mL) may be added.

At Step 177, the sample may be incubated at 56° C. for one hour. Thesample may be vortexed and pulse-spinned after incubation.

At Step 178, the sample may be incubated at 90° C. for one hour.

At Step 179, the sample may then be cooled to room temperature (e.g. for5 min).

At Step 180, RNAse A (e.g. 10 mg/mL) may optionally be added.

At Step 181, the sample may be incubated at 25° C. for 5 min. The samplemay be vortexed and pulse-spinned after incubation.

At Step 182, a sample lubricant (e.g. MOPS) may optionally be added tothe sample.

At Step 183, the sample may optionally be frozen for use at a laterdate.

At Step 184, the sample may be warmed to 20° C.

At Step 185, a nucleic acid dye or stain may optionally be added to thesample lysate for visualization and detection (e.g., for quantitation ofnucleic acid mass) of nucleic acid. For example, the addition of anucleic acid stain, such as a nucleic acid binding or intercalating dye,may be used when in-line quantitation of nucleic acid mass is performedduring the ITP process.

At Step 186, the sample may be loaded into one or more samplereservoirs. Prior to loading, the sample may be pulse vortexed (e.g.twice) and pulse-spinned to agitate and mix the sample. The ITP channelmay be pre-primed with leading electrolyte buffer, trailing electrolytebuffer, and/or other buffers as described herein. Alternatively, thesample may be loaded at the same time as the rest of the liquids in thechannel.

At Step 187, ITP may be performed. During the ITP process, the DNAwithin the sample may be purified (i.e. concentrated) as it movesthrough the channel until it reaches the elution reservoir as describedherein. The sample DNA may be quantified as described herein.

Also at Step 187, the eluted sample can may recovered by elution (e.g.by pipetting) from the elution reservoir.

FIG. 1B shows an exemplary process workflow for automated ITP. At step110, a protocol can be selected, such as by using a graphical userinterface on a benchtop device. The user interface software can enableease of use or hands-free operation. For example, a user can select froma menu (e.g., drop-down menu). Alternatively, the device can scan abarcode (e.g., optical barcode, RFID chip) associated with a sample or afluidic device chip which can indicate the protocol to be performed. Atstep 111, the instrument lid can be opened (e.g., manually orautomatically via motor). Motorized lid opening can be compatible withrobotic laboratory automation. At step 112, the user can load a chip(e.g., fluidic device) onto the benchtop instrument. The chip cancomprise a monolithic, multichannel SLAS standard microtiter plate (MTP)footprint for automated ITP. At step 113, ITP liquids can be loaded intothe chip wells. Reservoirs for ITP fluids and user samples can bedesigned for ease of loading, such as via a multichannel pipet (e.g., 9mm pitch SLAS standard microtiter plate format). Geometrical designs(e.g., capillary barriers) of the channels connecting reservoirs to theITP channel can resist gravity-driven flow or wetting of liquids intothe channel prior to operation. These structures can stop fluids indefined places within the ITP channel, including establishing theleading electrolyte/trailing electrolyte interface, as well as enablebubble-free loading. In some cases, prior to operation, pneumaticactuation can be applied to prime the channel. Chip material can beselected to prevent or resist wetting or wicking of fluids into channels(e.g., plastic with hydrophobic properties or a high contact angle). Theuser can load ITP reagents and buffers onto the chip (e.g., 5 differentfluids); alternatively, the chip can be provided with reagentspreloaded. At step 114, the user or the device can close the device lid.Sample loading can be actuated through gas or air ports on the chip.Wetting and/or gravity-driven flow can be used to fill channels withliquids, for example without active pressure application.

At step 115, the instrument can apply pressure to load fluids in thechip to prime the channels. At step 116, the device can check that thechannels have been appropriately primed. For example, optical (e.g.,reflectance), electrical, pressure, and/or flow rate sensors can be usedto check that fluids have been loaded to the correct locations withinthe chip. Sensors and device software can enable real time monitoringand control of liquid loading. ITP reagent and buffer loading can beconducted prior to loading sample onto the chip, so that in case ofmis-loading, sample material is not wasted. If the channels are notappropriately primed, the device can perform error reporting 130. Atstep 117, the device lid can be opened. At step 118, the sample can beloaded onto the device. Sample loading can be performed manually by auser, or can be performed in an automated manner, such as via laboratoryautomation robotics. Other sample preparation steps can also beconducted. For example, a paraffin-embedded sample (e.g., FFPE) can beloaded, and then the device can control the temperature within thesample reservoir to deparaffinize the sample. At step 119, the devicelid can be closed. At step 120, the device can perform a self-test. Forexample, electrical feedback from device electrodes interfacing withon-chip reservoirs can be used to self-test for successful priming ofliquids (e.g., bubble detection). Optical sensors can be used to enablefeedback on liquid priming status (e.g., whether or not a liquid hasreached a designated capillary barrier). Other sensing mechanisms, suchas those disclosed herein, can also be used. If the self-test determinesthat the device is not properly primed, the device can perform errorreporting 131.

At step 121, ITP-based purification can be conducted. Feedback controland process timing using sensors (e.g. triggering) as described hereincan be used to control and/or automate the ITP purification. The devicecan determine whether purification was successfully performed, and ifnot, the device can perform error reporting 132. At step 122, sensors onthe device (e.g., optical sensors) can be used to quantitate thesamples, for example by fluorescence, UV, or other optical detection.Sample sizing can also be performed. If the device determines that thesample was not properly quantitated or discovers other issues, thedevice can perform error reporting 133. At step 123, a conductivitychange can be detected, which can be used to indicate timing for endingthe ITP run (e.g., when the nucleic acids reach a designated elutionlocation or reservoir). Other detection methods described herein, suchas temperature or driving voltage, can also be used to determine end ofrun timing or other triggers. For example, a temperature or voltagesensor may be used to control an electric field applied to a channelwithin the device in order to automate the ITP process. As an example,an electric field may be applied to a channel to begin ITP purification.A sensed change in voltage may be used to trigger the start oftemperature or other sensing at a fixed location within the channel suchas at or near the elution reservoir. The voltage may change as the ITPzone comprising confined nucleic acids moves. Changes indicative of theITP zone passing through channel features such as a section of decreasedcross-sectional area may be sensed by a voltage sensor and feedback maybe used to alter the electric field, for example by reducing the appliedcurrent. A change in temperature may be detected as the ITP zone passesa temperature sensor at or near the elution reservoir and feedback fromthe sensor may be used to control the electric field, for example byremoving it to end the ITP run. At step 124, the device can terminatethe run, for example based on a trigger signal. The nucleic acids may bepositioned or isolated within the elution reservoir or region when theITP run is terminated. At step 125, the device can close the channels,which can fix the elution volume to maintain a constant volume for theelution (e.g., by resisting or preventing flow into the elutionreservoir or outlet reservoir during pipetting out of the elutedvolume). Fixing the elution volume can aid ease of use and can help forreporting the concentration of the eluted sample material. At step 126,the device lid can be opened (e.g., by a user or automatically).

At step 127, purified samples can be extracted from the device. Chipsand/or devices can be designed for a given elution volume, as discussedherein. Retrieval of purified material from the device can be performedvia pipetting or otherwise removing the material from the chip.Alternatively, sample extraction can be performed by interfacing the ITPchip with another fluidic chip or system (e.g., in the absence of anelution reservoir). Other fluidic systems can then be used to performother operations on the purified sample material, such as nextgeneration sequencing (NGS) library preparation, sample analysis such asPCR, ddPCR, other sequencing operations, or other downstream processes.At step 128, the device can report quantitative data about the sample,such as sample amount and/or sample concentration. The device cancontain an algorithm or other software for converting a measurement(e.g., a fluorescence signal) into a sample quantitation, and can reportthat data to a user. At step 129, the process ends.

FIG. 33 shows a detailed, non-limiting exemplary process workflow forautomated ITP using the methods, devices, and systems described herein.

At Step 250, the user may turn the instrument on.

At Step 251, instrument initialization may optionally be performed.Initialization may for example include one or more of the followingchecks/steps: (a) the pressure control tolerance may be checked at 0 psiand −0.1 psi; (b) a negligible flow rate within the channels withnegative pressure valves closed may be confirmed; (c) the proportionalvalve value may be observed; (d) the HVS temperature may be checked; (e)the HVS voltage rails may be checked; (f) no voltage with HVS disabledmay be confirmed; (g) the optics temperature may be checked; (h) theoptics voltage rails may be checked; (i) increases in pickoff value whenturning on each LED may be confirmed; (j) increases detector value whenturning on each LED may be confirmed; and/or (k) infrared sensors at ornear room temperature may be confirmed.

At Step 252, the instrument may optionally display a “Start” or “Home”menu to the user.

At Step 253, the user may optionally hit “Start New Run” on theinstrument display.

At Step 254, the instrument door may open.

At Step 255, the user may place a fresh microfluidic chip on theinstrument stage.

At Step 256, a “chip-in-place” sensor may optionally be used to detectif the chip has been placed on the instrument stage and/or if the chiphas been correctly placed or oriented on the stage.

At Step 257, the instrument may optionally read a bar code identifier onthe chip. For example, the user may use a handheld scanner.

At Step 258, the instrument may optionally select a protocol to runbased on the bar code identifier scanned.

At Step 259, the instrument may optionally check the temperature of theinstrument (e.g. of a heater). For example, the temperature of theheating/cooling element may be read by the instrument. The temperaturedetected by an optional thermal sensor may be read by the instrument.The temperature range of the instrument may be confirmed, for example bychecking that the instrument temperature and the temperature detected bythe thermal sensor are different by no more than about 5° C.

At Step 260, the instrument may optionally display instructions for howto load the buffers (e.g. one or more trailing electrolyte buffer, oneor more leading electrolyte buffer, one or more elution buffer, etc.)onto the chip. For example, the instrument may display visual and/orcolor-coded instructions to the user (e.g. as shown in FIG. 60).

At Step 261, the user may pipette the buffers onto the chip.

At Step 262, the user may close the instrument door, for example bypushing a button on the display.

At Step 263, the instrument may load one or more of the bufferingliquids into the chip. The display may optionally provide the user withan indication that loading is occurring, for example by displaying a“priming in progress” message.

At Step 264, the instrument may optionally check to make sure that thebuffering reagents have been loaded and that the channels have beencorrectly primed with the buffers. Loading may for example be confirmedby testing the electrical conductivity between two high voltageelectrodes as described herein. For example, 10 μA may be sourced fromone electrode and 10 μA may be sunk from another electrode. The voltagedifference across the two electrodes may be measured as describedherein. Priming of the channels may be confirmed prior to, during, orafter confirmation of buffer loading. Channel priming may for example beconfirmed by ensuring electrical conductivity between a source electrodeand a grounding electrode.

At Step 265, the instrument may open the door/lid to allow the user toaccess the chip following confirmation that the one or more buffers werecorrectly loaded.

At Step 266, the instrument may optionally display instructions to theuser on how to load the sample into the chip.

At Step 267, the user may pipette the sample(s) onto the chip. The usermay optionally remove a seal from the sample well(s) of the chip toenable sample loading. The user may optionally add an additional volumeof “topper” to the sample reservoir after loading the sample asdescribed herein.

At Step 268, the user may close the instrument door, for example bypushing a button on the display.

At Step 269, the instrument may optionally confirm that the sample hasbeen correctly loaded. Loading of the sample may be confirmed bychecking the electrical conductivity of each electrode to ground asdescribed herein. When one or more buffers remains unloaded after sampleloading, the instrument may load the remaining buffers onto the chip.

At Step 270, the instrument may begin the ITP run. The instrument mayoptionally wait a pre-determined amount of time after loading the chipto allow the fluids in the chip to equilibrate. Beginning the ITP runmay entail activating a high-voltage (“HV”) power supply, adjusting thetemperature of the chip to a run temperature (“T_run”), and/or turningon an optical detection system (e.g. a light emitting dioder). The runtemperature for a typical ITP procedure may for example be within arange of about 15° C. to about 23° C.

At Step 271, the instrument may optionally record and process voltagesignals detected as described herein.

At Step 272, the instrument may optionally detect a change(s) in voltageto act as a trigger to begin ITP as described herein. A change involtage may optionally act as a trigger to alter the drivingelectrode(s) polarity and/or driving voltage as described herein.

At Step 273, the instrument may optionally perform optical detection.

At Step 274, the instrument may optionally process the detected opticalsignals.

At Step 275, the instrument may optionally sense a change in temperatureat a pre-determined location within the chip, for example using aninfrared sensor as described herein. A change in temperature mayoptionally act as a trigger to end ITP as described herein.

At Step 276, the instrument may optionally detect a change(s) in voltageto act as a trigger to end ITP as described herein.

At Step 277, the instrument may optionally be triggered to shut off thehigh-voltage power supply, thereby ending the ITP run. The instrumentmay also shut down the optical system.

At Step 278, the instrument may optionally close off the channels usinga channel closer as described herein.

At Step 279, the instrument may optionally display an indicator ormessage to the user to alert them that the ITP has been completed.

At Step 280, the instrument may optionally display any quantitative datacollected during the ITP run to the user.

At Step 281, the user may return or be present at the machine.

At Step 282, the instrument may optionally hold the chip at a fixedtemperature until the user returns to the instrument as in Step 281. Thefixed temperature may for example be within a range of about 4° C. toabout 20° C.

At Step 283, the user may optionally open the instrument door/lid, forexample by pushing a button on the display.

At Step 284, the user may optionally recover the sample from the elutionreservoir.

Alternatively or in combination, the instrument may optionally recoverthe sample from the elution reservoir automatically. The sample mayoptionally then be used for further downstream assays as desired by theuser.

At Step 285, the user may user may remove the used chip from theinstrument.

At Step 286, the chip-in-place sensor may optionally detect removal ofthe chip by the user. The bar code information stored on the instrumentmay be cleared.

At Step 287, the user may optionally ready their reagents and samplesfor additional ITP runs with a new chip if desired.

At Step 288, the user may close the door.

At Step 289, the user may optionally repeat Steps 253 to 288 with a newchip, buffers, samples, etc. as many times as desired. The instrumentmay optionally adjust the temperature of the instrument while idlingbetween runs, for example to a temperature within a range of about 20°C. to about 25° C.

At Step 290, the user may optionally transfer data collected during theITP run(s), for example via a USB port on the instrument or via awireless connection.

At Step 291, the user may optionally turn the instrument off. In someinstances, the instrument may be programmed to turn off following apre-determined amount of idle time when the chip-in-place sensorconfirms that there are no chips in the instrument.

Table 1 shows typical operating times for various steps, manual(performed by user) or automated (performed by instrument) of the ITPprocess using the exemplary methods described in FIG. 33.

TABLE 1 Step: Action Value Step 261: User Loads Buffers Time =<10 minStep 263: Priming Time =<20 min Step 267: User Pipettes Sample Time =<10min Steps 272-274: Run time, from start to optics 10-90 min window Steps275-276: Run time between optical detect and 5-20 min thermal event (IRsensor detect) Step 277: Remaining run time after thermal event 1-2 min(IR sensor detect) Step 278: Time to close channels after HV shutoff =<5min Step 282: Hold Time >=5 min Step 283: Idle time before next run =<5min Step 284: User unloads samples =<10 min

These issues can be especially important to address for precious,difficult to collect, or low-abundance (e.g., less than 100 ng ofnucleic acid or samples containing a low abundance of undamaged oruncrosslinked nucleic acids) samples. For such samples, currentprotocols may lack repeatability, introduce loss of sample material,introduce bias for short or long nucleic acid targets, introduce biastowards sequence of nucleic acid targets, and/or lack repeatability.Such protocols may also lack compatibility with process automation ordownstream analyses. Current protocols for nucleic acid preparation caninclude liquid phase extraction (LPE) such as phenol-chloroformextraction or Trizol extraction, and solid phase extraction (SPE). SPEtype approaches can use structures including packed beads, monolithicporous structures, and/or magnetic beads. In some cases, LPE and SPEtype approaches can lead to mechanical shearing during processing whichcan cause fragmentation and/or reduce the yield of long or highmolecular weight nucleic acids.

The isotachophoresis methods and devices provided herein are especiallywell-suited to performing extraction of nucleic acids from lysates ofsolid or semi-solid tissues. Solid phase extraction (SPE) techniquestypically process lysates by pumping the entire lysate sample volumethrough a column in order to selectively adsorb nucleic acids onto thesurfaces of the column. Such pumping of a complex lysate, which maycomprise a liquid-particle mixture, through a porous column can resultin clogging or fouling of the column which can reduce the efficiency ofnucleic acid extraction. In contrast, the isotachophoresis methods anddevices described herein often do not involve pumping or “filtering” theentire lysate sample volume through a column. Instead, an electric fieldmay be applied to the lysate in order to cause the charged, solvatednucleic acids dispersed throughout the complex sample lysate to migratethrough and out of the continuous liquid phase of the sample. Nucleicacids may comprise a relatively high electrophoretic mobility magnituderelative to other solutes, debris, or contaminants in the sample lysate.Solutes in the sample may have a relatively low electrophoretic mobilityand be too low to focus into the isotachophoresis zone located at theinterface between the leading electrolytes and trailing electrolytes.Application of an electric field may cause the nucleic acids to migratewhile particles and/or other tissue debris (including for example celldebris, unlysed cells, or tissue which may connect cells to other cells)are left behind. The isotachophoresis methods and devices providedherein therefore can be well-suited to extract the charged, solvatednucleic acids out of the complex lysed solid tissue samples withouthaving to process the entire mixture through a column as in SPE.

As used herein, “particles” may refer to components of a sample mixtureor a sample lysate mixture which are a different phase than thecontinuous liquid phase of the sample (e.g., an aqueous solution).Particles may be non-liquid components of the sample mixture. Particlescan be, for example, suspended solid particles or colloidal bodiessuspended within a sample. Such particles can have a variety ofcharacteristic length scales ranging from about 1 nanometer (nm) toabout 1 millimeter (mm). In some instances, particles may not besingle-celled organisms or cells.

The isotachophoresis methods and devices provided herein may provide forreduced rates of strain as the sample moves through the channel comparedto typical SPE methods. In some cases, the methods and devices providedherein have rates of strain of less than about 250 s⁻¹, 500 s⁻¹, 750s⁻¹, 1000 s⁻¹, 2000 s⁻¹, 3000 s⁻¹, 4000 s⁻¹, 5000 s⁻¹, 6000 s⁻¹, 7000s⁻¹, 8000 s⁻¹, 9000 s⁻¹, or 10,000 s⁻¹. In some cases, the methods anddevices provided herein have rates of strain of more than about 250 s⁻¹,500 s⁻¹, 750 s⁻¹, 1000 s⁻¹, 2000 s⁻¹, 3000 s⁻¹, 4000 s⁻¹, 5000 s⁻¹, 6000s⁻¹, 7000 s⁻¹, 8000 s⁻¹, 9000 s⁻¹, or 10,000 s⁻¹. In some cases, themethods provided herein may be performed without centrifugation.

Isotachophoresis Chemistry and Operation

FIG. 2A shows an exemplary schematic of an isotachophoresis (ITP)process purifying nucleic acid. A sample 201, for example a lysed solidtissue sample, comprising nucleic acids (DNA and RNA) 202 andcontaminants 203 may be loaded with trailing electrolytes (TE) 204 intoan isotachophoresis channel 200 containing leading electrolytes (LE)205. Under the influence of an electric field 220 applied to theisotachophoresis channel 210, the nucleic acids 212 may migrate awayfrom the contaminants 213. The electric field may also cause thetrailing electrolytes 214 to migrate through the channel in a positionthat is generally behind the nucleic acids, and generally causes theleading electrolytes 215 to migrate through the channel generally aheadof the nucleic acids. The magnitude of the effective mobility of theleading electrolytes is greater than the magnitude of the effectivemobility of the nucleic acids, which in turn is greater than themagnitude of the effective mobility of the trailing electrolytes, whichis greater than the magnitude of the effective mobility of thecontaminants.

FIG. 2B shows an exemplary schematic of a process to de-crosslinknucleic acids while separating de-crosslinked nucleic acids fromcrosslinked nucleic acids and contaminants (e.g., paraffin) usingisotachophoresis (ITP) on a fluidic device. In some instances, thecontaminants may comprise the crosslinked nucleic acids. Aparaffin-embedded sample may be loaded onto the fluidic device in analkaline buffer and incubated for 10-30 minutes at about pH 10 and atemperature from about 50° C. to about 80° C. for tissue lysis andinitial deparaffinization. Incubation may occur prior to or whileapplying an electric field to perform isotachophoresis. Alternatively,the sample can be loaded in a leading electrolyte buffer. Afterincubation, at a first time point 240, the sample comprising crosslinkednucleic acids 236 and paraffin 237 may be located in an ITP channel withtrailing electrolytes 232. Ahead in the ITP channel, in a leadingelectrolyte (LE) zone 238 are leading electrolytes 231 and Proteinase Kenzymes 233. At a second time point 250, at 50° C., ITP-driven pHquenching reduces the pH, and Proteinase K enzymes are contacting andde-crosslinking the crosslinked nucleic acids, producing non-crosslinkednucleic acids 235 which focus at in the ITP zone 239 between thetrailing electrolytes and leading electrolytes. Reduction of pH (e.g. toa range from about 10-12 to about 7 (or from about 6.5 to about 8.5))can provide an environment appropriate for enzymatic activity andimproved chemical stability of nucleic acids. At a third time point 260the Proteinase K has de-crosslinked more nucleic acids, resulting infree protein 234, and the de-crosslinked nucleic acids have furthermigrated upstream from the paraffin, free protein, and othercontaminants. The operation of such a process can be conductedautomatically by the fluidic device or by a benchtop system.

In some cases, the sample may be loaded in a sample buffer comprising aconcentration of leading electrolytes 205, 231 that differs from theconcentration of leading electrolytes 205, 231 used to performisotachophoresis. In some cases, the sample may be loaded in a samplebuffer comprising a second leading electrolyte which differs from theleading electrolyte 215. The second leading electrolyte can have aneffective mobility magnitude greater than the magnitude the effectivemobility of the nucleic acid. The second leading electrolyte can have aneffective mobility magnitude less than the effective mobility magnitudeof the leading electrolyte 215.

In some cases, a pH of the sample may be quenched by conductingisotachophoresis. In some instances, the pH of the sample may bequenched within a range of about 6.5 to about 8.5, for example about 7or 7.5.

Various leading electrolytes and trailing electrolytes can be used toconduct ITP. Leading electrolytes can be selected to have a greatereffective mobility magnitude than the extraction target (e.g., nucleicacids), and trailing electrolytes can be selected to have a lessereffective mobility magnitude than the extraction target. Leading and/ortrailing electrolytes can be present at a concentration from about 10 mMto about 200 mM. Leading and/or trailing electrolytes can be present ata concentration of about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160mM, 170 mM, 180 mM, 190 mM, or 200 mM. Leading and/or trailingelectrolytes can be present at a concentration of at least about 10 mM,20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM,120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200mM. Leading and/or trailing electrolytes can be present at aconcentration of at most about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM,70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160mM, 170 mM, 180 mM, 190 mM, or 200 mM. Leading electrolytes used in aparticular instance of ITP can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more different ion species. Trailing electrolytes used in aparticular instance of ITP can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more different ion species. Different species of ions in the leadingelectrolytes and/or trailing electrolytes can be present at differentconcentrations. Different concentrations of ions, such as within thetrailing electrolytes or the leading electrolytes, can be selected tomanipulate the size of a spacing zone. The spacing zone can be used tofurther separate one type of target from another, such as separatingdecrosslinked from protein crosslinked nucleic acids.

The trailing electrolytes can comprise a mixture of ions with differentmagnitudes of effective mobilities. Use of a first trailing electrolyteion with a first effective mobility magnitude and a second trailingelectrolyte ion with a second effective mobility magnitude lower thanthat of the first ion can be used to separate non-crosslinked nucleicacids from protein crosslinked nucleic acids, while separating both (orat least the decrosslinked nucleic acids) from contaminants. In such acase, the non-crosslinked nucleic acids can have a greater effectivemobility magnitude than the first trailing electrolyte ions, which canhave a greater effective mobility magnitude than the crosslinked nucleicacids, which in turn can have a greater effective mobility magnitudethan the second trailing electrolyte ions, which in turn can have agreater effective mobility magnitude than the contaminants. For example,crosslinked and non-crosslinked nucleic acids can be enriched separatelyby conducting isotachophoresis using a leading electrolyte and twotrailing electrolytes, such as caproic acid as the first ion and HEPESas the second ion.

Electrolyte ions can also be selected based on acidity (e.g., pKa). Ionswith particular pKa can be selected, for example, to effect a pH changealong an ITP channel. Ions can also be selected for non-electrophoreticreasons, such as compatibility with downstream processes (e.g.,enzymatic processes such as PCR or next-generation sequencing librarypreparation). For example, caproic acid, MOPS, and HEPES can be selectedfor good downstream enzymatic compatibility.

Exemplary leading electrolyte ions include but are not limited tohydrochloric acid, acetic acid, 2-chloroisocrotonic acid, salicylicacid, chlorocrotonic acid, nicotinic acid, gallic acid, trichlorolacticacid, butyric acid, sulfanilic acid, benzoic acid, crotonic acid,trichloroacrylic acid, propionic acid, levulinic acid, sorbic acid,orotic acid, valeric acid, picric acid, 2-naphtalenesulfonic acid,saccharin, dinitrophenol, p-toluenesulfonic acid, aspartic acid,trimethylacrylic acid, isocaproic acid, caproic acid, octylsulfonicacid, nitrophenol, GABA, cacodylic acid, trimetylpyruvic acid,ethylmaleic acid, ethylfumaric acid, toluic acid, enanthylic acid,mandelic acid, cinnamic acid, cresol, glutamic acid, MES, isomersthereof, and combinations thereof.

Exemplary trailing electrolyte ions include but are not limited tocaprylic acid, gluconic acid, vanillic acid, decylsulfonic acid,aspirin, glucuronic acid, pelargonic acid, benzylasparatic acid,ascorbic acid, dodecylsulfonic acid, MOPS(3-(N-morpholino)propanesulfonic acid), dichlorophenol, caproic acid,capric acid, tyrosine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), isomers thereof,and combinations thereof.

Use of a mixture of different trailing electrolyte ions can be used toachieve mobility bracketed separations (e.g., separation ofnon-crosslinked nucleic acids from crosslinked nucleic acids fromcontaminants), compatibility with downstream assays, favorable surfaceenergy or contact angles between fluids and fluidic device materials,buffering capacity, and total ion solubility.

Leading electrolytes may be loaded in a leading electrolyte buffer. Theleading electrolyte buffer may comprise one or more leading electrolytesto compact the target nucleic acids behind the leading electrolytes inthe channel during ITP. The leading electrolytes may also separate thetarget nucleic acids from any contaminants or inhibitors which have aneffective mobility magnitude greater than that of the target nucleicacids. The leading electrolyte buffer may, for example, comprisechloride. The leading electrolyte buffer may comprise a sufficientconcentration of chloride such that the separation capacity of theleading electrolyte buffer is greater than the ionic strength of thesample nucleic acids. The leading electrolyte buffer may comprise a pHcompatible with nucleic acid stability. The leading electrolyte buffermay comprise a surfactant (e.g. Tween) in order to reduce or minimizeelectroosmotic flow. The leading electrolyte buffer may comprise asurfactant (e.g. Brij-35) in order to reduce or minimize surfaceadsorption. The leading electrolyte buffer may comprise one or moresurfactants as described herein. The concentration of the one or moresurfactants may be adjusted so as to ensure that the LE does notuncontrollably wet past the capillary barriers (e.g. the plateaucapillary barriers) described herein.

In some embodiments, leading electrolytes may be loaded in a highconcentration leading electrolyte buffer, which may act to buffer alower concentration leading electrolyte buffer. The high concentrationleading electrolyte buffer may have sufficient buffering capacity so asto not change pH during the electrolysis process that occurs throughoutthe ITP run. The high concentration leading electrolyte buffer maycomprise one or more leading electrolytes, which may be the same as theleading electrolytes of the leading electrolyte buffer but at a higherconcentration. The high concentration leading electrolyte buffer maycomprise Tris and chloride. The high concentration leading electrolytebuffer may comprise Tris and chloride at a ratio configured to maximizebuffering capacity, for example a high Tris:chloride ratio to provide asource of Tris during the ITP run. The high concentration leadingelectrolyte buffer may comprise one or more surfactant in order toensure that the high concentration leading electrolyte buffer wets thewalls of the high concentration leading electrolyte buffer reservoir andloads to the capillary barriers upon application of negative pressure.The concentration of the one or more surfactants may be adjusted so asto ensure that the LE does not uncontrollably wet past the capillarybarriers (e.g. the ramp barriers) described herein.

In some embodiments, leading electrolytes may be loaded in an elutionbuffer. The elution buffer may comprise one or more leadingelectrolytes. The one or more leading electrolytes of the elution buffermay have a lower ionic strength than the leading electrolytes of theleading electrolyte buffer. The one or more leading electrolytes mayenable a hand-off of the ITP band from the higher ionic strength leadingelectrolyte buffer to the lower ionic strength elution buffer. Theelution buffer may provide compatibility with one or more downstreamassays as described herein (e.g. NGS library prep, PCR, etc.). Theelution buffer may comprise Tris and chloride, for example 10 mMTris-HCl. The elution buffer may comprise a pH compatible with nucleicacid stability. The leading electrolyte buffer may comprise a surfactant(e.g. Tween) in order to reduce or minimize electroosmotic flow. Theleading electrolyte buffer may comprise a surfactant in order to reduceor minimize bubble growth in the fluidic channel (e.g. duringtemperature measurements as described herein). The leading electrolytebuffer may comprise a surfactant in order to reduce or minimize surfaceadsorption. The elution buffer may comprise one or more surfactants asdescribed herein. The concentration of the one or more surfactants maybe adjusted so as to ensure that the elution buffer does notuncontrollably wet past the capillary barriers (e.g. the ramp barriers)described herein.

In some embodiments, leading electrolytes may be loaded in a highconcentration elution buffer, which may act to buffer a lowerconcentration elution buffer (e.g. a lower concentration suitable forextraction and use in downstream assays as described herein). The highconcentration elution buffer may have sufficient buffering capacity soas to not change pH during the electrolysis process that occursthroughout the ITP run. The high concentration elution buffer may have aminimal (e.g. less than 1 μl) amount of carryover between the highconcentration elution buffer and the elution buffer. The highconcentration elution buffer may comprise a sufficiently low ionconcentration such that this carryover does not impact downstreamcompatibility. The high concentration elution buffer may comprise Trisand chloride. The high concentration elution buffer may comprise Trisand chloride at a ratio configured to maximize buffering capacity, forexample a high Tris:chloride ratio to provide a source of Tris duringthe ITP run. The high concentration elution buffer may comprise Tris andchloride at a sufficiently high ion concentration to achieve robustbuffering while minimizing the impact of carryover. The highconcentration elution buffer may comprise one or more surfactant inorder to ensure that the high concentration elution buffer wets thewalls of the high concentration elution buffer reservoir and loads tothe capillary barriers upon application of negative pressure. Theconcentration of the one or more surfactants may be adjusted so as toensure that the high concentration elution buffer does notuncontrollably wet past the capillary barriers (e.g. the ramp barriers)described herein.

Trailing electrolyte may be in a trailing electrolyte buffer. Thetrailing electrolyte buffer may comprise one or more trailingelectrolytes compact the target nucleic acids in front of the trailingelectrolytes in the channel during ITP. The trailing electrolytes mayalso separate the target nucleic acids from any contaminants orinhibitors which have an effective mobility magnitude less than that ofthe target nucleic acids. The trailing electrolyte buffer may havesufficient buffering capacity so as to not change pH during theelectrolysis process that occurs throughout the ITP run. The trailingelectrolyte buffer may, for example, comprise caproic acid. The trailingelectrolyte buffer may comprise MOPS. The trailing electrolyte buffermay comprise caproic acid and MOPS. The trailing electrolyte buffer maycomprise a high concentration of caproic acid. A high concentration ofcaproic acid may lead to an overly wetting fluid. MOPS may be added tothe caproic acid of the trailing electrolyte buffer to provide thenecessary buffering capacity in the trailing electrolyte reservoirwithout the increased wetting of too high a concentration of caproicacid. The trailing electrolyte buffer may comprise a pH compatible withnucleic acid stability.

The leading electrolyte buffer may comprise one or more surfactants(e.g. Tween) as described herein. The concentration of the one or moresurfactants may be adjusted so as to ensure that the TE does notuncontrollably wet past the capillary barriers (e.g. the plateaucapillary barriers) described herein. The concentration of the one ormore surfactants may be adjusted to create no or small bubbles duringthe electrolysis process, as opposed to large bubbles, which may lead tofluid fluctuations that can cause disturbance of the ITP band, thevoltage trace, and/or the temperature trace, or very large bubbles,which may move and negatively impact triggering.

Isotachophoresis can quench the pH of a sample to neutral or aboutneutral. Ions affecting the local pH (e.g., sodium ions (Na+)) can bedisplaced from the sample zone during isotachophoresis, thereby shiftingthe pH in the sample zone toward neutral.

Isotachophoresis can be conducted at a range of voltages, currents, andfield strengths. For example, isotachophoresis can be conducted at avoltage from about 100 V and about 1500 V. Isotachophoresis can beconducted at a voltage of about 100 V, 200 V, 300 V, 400 V, 500 V, 600V, 700 V, 800 V, 900 V, 1000 V, 1100 V, 1200 V, 1300 V, 1400 V, or 15000V. Isotachophoresis can be conducted at a voltage of at least about 100V, 200 V, 300 V, 400 V, 500 V, 600 V, 700 V, 800 V, 900 V, 1000 V, 1100V, 1200 V, 1300 V, 1400 V, or 15000 V. Isotachophoresis can be conductedat a voltage of at most about 100 V, 200 V, 300 V, 400 V, 500 V, 600 V,700 V, 800 V, 900 V, 1000 V, 1100 V, 1200 V, 1300 V, 1400 V, or 15000 V.Isotachophoresis can be conducted at a current from about 10 nA to about10 mA. Isotachophoresis can be conducted at a current of about 10 nA, 20nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA, or 10 mA. Isotachophoresis can beconducted at a current of at least about 10 nA, 20 nA, 30 nA, 40 nA, 50nA, 60 nA, 70 nA, 80 nA, 90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA,600 nA, 700 nA, 800 nA, 900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7mA, 8 mA, 9 mA, or 10 mA. Isotachophoresis can be conducted at a currentof at most about 10 nA, 20 nA, 30 nA, 40 nA, 50 nA, 60 nA, 70 nA, 80 nA,90 nA, 100 nA, 200 nA, 300 nA, 400 nA, 500 nA, 600 nA, 700 nA, 800 nA,900 nA, 1 mA, 2 mA, 3 mA, 4 mA, 5 mA, 6 mA, 7 mA, 8 mA, 9 mA, or 10 mA.Isotachophoresis can be conducted at a field strength of from about 10V/cm to about 100 V/cm. Isotachophoresis can be conducted at a fieldstrength of about 10 V/cm, 15 V/cm, 20 V/cm, 25 V/cm, 30 V/cm, 35 V/cm,40 V/cm, 45 V/cm, 50 V/cm, 55 V/cm, 60 V/cm, 65 V/cm, 70 V/cm, 75 V/cm,80 V/cm, 85 V/cm, 90 V/cm, 95 V/cm, or 100 V/cm. Isotachophoresis can beconducted at a field strength of at least about 10 V/cm, 15 V/cm, 20V/cm, 25 V/cm, 30 V/cm, 35 V/cm, 40 V/cm, 45 V/cm, 50 V/cm, 55 V/cm, 60V/cm, 65 V/cm, 70 V/cm, 75 V/cm, 80 V/cm, 85 V/cm, 90 V/cm, 95 V/cm, or100 V/cm. Isotachophoresis can be conducted at a field strength of atmost about 10 V/cm, 15 V/cm, 20 V/cm, 25 V/cm, 30 V/cm, 35 V/cm, 40V/cm, 45 V/cm, 50 V/cm, 55 V/cm, 60 V/cm, 65 V/cm, 70 V/cm, 75 V/cm, 80V/cm, 85 V/cm, 90 V/cm, 95 V/cm, or 100 V/cm.

Isotachophoresis can be used to concentrate nucleic acids in a sample.The concentration of nucleic acids in a sample can be increased afterisotachophoresis by at least about 2-fold, 5-fold, 10-fold, 100-fold,1,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold,100,000,000-fold, or 1,000,000,000-fold. The operation time forconcentration of nucleic acids with isotachophoresis can be less than orequal to about 5 hours, 4.5 hours, 4 hours, 3.5 hours, 3 hours, 2.5hours, 2 hours, 1.5 hours, 1 hours, 50 minutes, 40 minutes, 30 minutes,20 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30seconds, 20 seconds 10 seconds, or 1 second. In some cases,isotachophoresis can be used to increase the concentration of nucleicacids in a sample by 1,000,000-fold in less than or equal to about 2minutes. In some cases (e.g., from a sample of 25 μL blood lysate),isotachophoresis can be used to increase the concentration of nucleicacids in a sample by 100,000-fold in less than or equal to about 5minutes.

Techniques of the present disclosure can be used to reduce theconcentration of crosslinked nucleic acids in a sample. Theconcentration of crosslinked nucleic acids in a sample can be reducedafter isotachophoresis by at least about 2-fold, 5-fold, 10-fold,100-fold, 1,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold,10,000,000-fold, 100,000,000-fold, or 1,000,000,000-fold.Isotachophoresis can be used to reduce the concentration of acontaminant in a sample. The concentration of contaminants in a samplecan be reduced after isotachophoresis by at least about 2-fold, 5-fold,10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold,1,000,000-fold, 10,000,000-fold, 100,000,000-fold, or1,000,000,000-fold.

Nucleic acid samples can contain from about 0.1 picograms (pg) to about25 micrograms (m). For example, nucleic acid samples can contain fromabout 5 pg to about 5 μg. Nucleic acid samples can contain about 0.1 pg,0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1 pg, 2pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 20 pg, 30 pg, 40pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg,500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 nanogram (ng), 2 ng, 3 ng, 4ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng,700 ng, 800 ng, 900 ng, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg,9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, or 25 μg.

Nucleic acid samples can comprise deoxyribonucleic acids (DNA),single-stranded DNA, double-stranded DNA, genomic DNA, complementaryDNA, ribonucleic acids (RNA), ribosomal RNA, transfer RNA, messengerRNA, micro RNA, or the like, or any combination thereof. Nucleic acidsamples can comprise a length of at least about 0.5, 1, 2, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or500 kB or more. Techniques of the present disclosure can be used toextract different sample types in different channels of a fluidicdevice. For example, different channels may be used to extract nucleicacids of different lengths and/or different types.

In some instances, a characteristic of a nucleic acid sample may becompared to one or more nucleic acids from another sample. Thecharacteristic may for example be an expression level, a nucleic acidsequence, a molecular weight, nucleic acid integrity, nucleic-acidstranded-ness, or nucleic acid purity.

Nucleic acid samples can be of a particular quality before and/or afterextraction or other processing. Nucleic acid quality can be assessed byvarious metrics, including but not limited to RNA integrity number(RIN), DNA integrity number (DIN), size distribution (e.g., usingelectrophoresis), and ability to be amplified (e.g., by PCR) orotherwise enzymatically processed (e.g. fragmentation, ligation,a-tailing, or hybridization for next generation sequencing librarypreparation). Techniques of the present disclosure can be used toextract or process nucleic acids and provide extracted or processednucleic acids with a RIN of at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or10.0. Techniques of the present disclosure can be used to extract orprocess nucleic acids and provide extracted or processed nucleic acidswith a RIN of at most about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0.Techniques of the present disclosure can be used to extract or processnucleic acids and provide extracted or processed nucleic acids with aDIN of at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. Techniques ofthe present disclosure can be used to extract or process nucleic acidsand provide extracted or processed nucleic acids with a DIN of at mostabout 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. Techniques of the presentdisclosure can be used to extract or process nucleic acids and provideextracted or processed nucleic acids such that at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99%of the mass of the nucleic acids of the sample has a molecular weight ofat least about 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, or 500 kB or more. In some cases,about 90% to about 100% of the mass of the processed nucleic acids arefrom about 10 to about 1000 bp, from about 200 to about 2000 bp, or fromabout 200-5000 bp.

Isotachophoresis can be used to extract nucleic acids at an extractionefficiency or yield, characterized as the percent yield of nucleic acidfrom a given starting amount of nucleic acid. Techniques of the presentdisclosure can provide extracted nucleic acids at a yield of at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9%. Techniques of thepresent disclosure can provide high yields even for low input amountsnucleic acid, including less than or equal to about 10⁴ nanograms (ng),10³ ng, 10² ng, 10¹ ng, 10⁰ ng, 10⁻¹ ng, or 10⁻² ng. FIG. 3, forexample, shows exemplary nucleic acid yields from a range of differentinput amounts and sources of nucleic acid. High yield and/or low loss ofnucleic acids can be important for next generation sequencing librarypreparations. Recovery of nucleic acids can be at or near 100%.

Techniques of the present disclosure can extract nucleic acids with lowor no sequence bias. That is, the sequence composition of the extractedand purified nucleic acids (e.g., ratio of GC-rich nucleic acids toAT-rich nucleic acids) can be similar to or the same as the sequencecomposition of the input nucleic acids (see, e.g., FIG. 4A). Thedifference in sequence composition of the extracted nucleic acids fromthe sequence composition of the input nucleic acids can be less than orequal to about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

Techniques of the present disclosure can extract nucleic acids with lowor no length bias. That is, the length distribution of the extractednucleic acids (e.g., the proportions of nucleic acids of differentsizes) can be similar to or the same as the length distribution of theinput nucleic acids (see, e.g., FIG. 4B). The difference in lengthdistribution of the extracted nucleic acids from the length distributionof the input nucleic acids can be less than or equal to about 20%, 15%,10%, 5%, 4%, 3%, 2%, or 1%. For example, short nucleic acids (e.g.,about 10 to about 300 bp), long nucleic acids (e.g., about 10 kB, 20 kB,30 kB, 40 kB, 50 kB, 60 kB, 70 kB, 80 kB, 90 kB, 100 kB, or greater), orboth short and long nucleic acids can be extracted with reduced drop outor bias. Solid phase columns can, in some cases, lose up to 100% ofshort and/or long nucleic acid material. Techniques of the currentdisclosure can recover nucleotides from single base to hundreds ofkilobases in size. Techniques of the present disclosure can recover atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, 96%,97%, 98%, 99%, 99.9%, or 100% of short and/or long nucleic acids presentin the sample.

Techniques of the present disclosure can result in the removal ofcontaminants from the sample. Contaminants can include but are notlimited to embedding material, cell debris, extracellular matrixcomponents, tissue debris, embedding debris, lipids, carbohydrates,enzymes, ligation by-products, primers, unbound probes or ligators,divalent metals, detergents, preservatives, fixatives, anti-coagulants,collagen fibers, and PCR inhibitors. Contaminants can originate from thetissue or cells of the sample, from preservatives or embedding materialsused on the sample, or from previous preparations, reactions, or assaysperformed on the sample. For example, enzymes such as restrictionnucleases can be used to prepare DNA for a fingerprinting assay, andsubsequent to digestion (e.g., DNase digestion), DNA can be separatedfrom the enzyme.

Samples

The techniques of the present disclosure can be used to processdifferent sample types, including but not limited to biological samples,solid tissue, biopsies, tissue biopsies, liquid biopsies, organs,tumors, fresh tissue, solid organs, preserved tissue (e.g., FFPE),dissected FFPE, fresh frozen tissue, fixed samples, fixed tissue,embedded samples, lysed samples, un-lysed samples, samples comprisingconnections between cells (e.g. gap junctions, tight junctions, adherentjunctions), samples comprising lysed solid tissue and nucleic acids,multiphasic samples, inhomogeneous liquids or solutions (such as tissue,whole blood, or unlysed cell suspensions), biological samples comprisinggenomic DNA, lysed and un-lysed whole blood, plasma and serum, buccalswabs, dried blood spots and other forensic samples, fresh or freshfrozen (FF) tissues, cultured or harvested cells (lysed and un-lysed)from blood or tissues, fixed cells, stool, and bodily fluids (e.g.,saliva, urine), or any combination thereof. Non-limiting examples ofsolid organs include liver, pancreas, brain, heart, gall bladder, colon,lung and reproductive organs. Samples can include cellular and cell-freenucleic acids, for both eukaryotic and prokaryotic organisms. Fixedsamples can be chemically fixed or physically fixed (e.g., heating orfreezing). For example, samples can be chemically fixed with a chemicalfixative such as formalin, neutral buffered formalin (NBF),formaldehyde, paraformaldehyde, glutaraldehyde, glyoxal, mercuricchloride, zinc salts, Bouin's fluid, alcohol-formalin-acetic acid (AFAor FAA), citrate-acetone-formalin (CAF), acetone, methanol, ethanol,Clarke's fluid, Carnoy's fluid, or Puchtler's methacarn. Embeddedsamples can be embedded in materials including but not limited to wax(e.g., paraffin), agar, gelatin, or plastic resins. Formalin-fixedparaffin-embedded (FFPE) samples can be processed using techniques ofthe present disclosure. Samples can comprise buccal swabs, blood spots,and other forensic samples. Samples can comprise clinical samples, fineneedle aspirates, biopsies, whole blood, lysed blood, serum, plasma,urine, cell culture lysate or freshly harvested cell (e.g., blood cell,dissociated fresh tissue, stem cell) lysate, blood cells, circulatingcells (e.g., circulating tumor cells (CTCs)), nucleic acids from bloodor other bodily fluid, and other sample categories. Cell-free nucleicacids (e.g., cfDNA or cfRNA) can be recovered, such as from wholeun-lysed blood, using techniques of the present disclosure; often thecell-free nucleic acids are circulating cell-free nucleic acids. Samplescan be from a variety of sources, including but not limited to normaltissue, benign neoplasms, malignant neoplasms, stem cells, human tissue,animal tissue, plant tissue, bacteria, viruses, and environmentalsources (e.g., water). Human or animal tissues can include but are notlimited to epithelial tissue, connective tissue (e.g., blood, bone),muscle tissue (e.g., smooth muscle, skeletal muscle, cardiac muscle),and nervous tissue (e.g., brain, spinal cord).

Samples can comprise one or more particles in suspension. The one ormore particles may range from colloidal size to visible. The one or moreparticles can have a size of at least about 1 nanometer (nm), 10 nm, 20nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800nm, 900 nm, 950 nm, 1 micrometer (μm), 10 μm, 20 μm, 30 μm, 40 μm, 50μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 175 μm, 200 μm, 225 μm,250 μm, 275 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm,650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, or 1 millimeter(mm). The one or more particles can have a size of at most about 1nanometer (nm), 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm,90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm,500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 950 nm, 1 micrometer (μm), 10μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 350 μm, 400 μm, 450μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900μm, 950 μm, or 1 millimeter (mm). The one or more particles can be thesame size or different sizes. A sample may for example comprise aplurality of particles ranging in size from 1 nm to 500 μm.

Samples of various volumes can be processed on a fluidic device (e.g.,to extract and purify nucleic acids). For example, a sample volume (withor without buffer) can be at least about 1 nanoliter (nL), 10 nL, 20 nL,50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL,40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 175 μL, 200μL, 225 μL, 250 μL, 275 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 600μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6mL, 7 mL, 8 mL, 9 mL, or 10 mL. A sample volume (with or without buffer)can be at most about 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL,70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7mL, 8 mL, 9 mL, or 10 mL. In some cases, a sample volume can be fromabout 1 nL to about 10 nL. A sample volume (with or without buffer) canbe at least about 1 nanoliter (nL), 10 nL, 20 nL, 50 nL, 100 nL, 200 nL,500 nL, 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7mL, 8 mL, 9 mL, or 10 mL. In some cases, a sample volume can be fromabout 1 nL to about 10 nL.

Samples with different numbers of cells can be processed on a fluidicdevice (e.g., to extract and purify nucleic acids). For example, asample can contain less than or equal to about 20,000 cells, 15,000cells, 10,000 cells, 9,000 cells, 8,000 cells, 7,000 cells, 6,000 cells,5,000 cells, 4,500 cells, 4,000 cells, 3,500 cells, 3,000 cells, 2,500cells, 2,000 cells, 1,500 cells, 1,000 cells, 900 cells, 800 cells, 700cells, 600 cells, 500 cells, 400 cells, 300 cells, 200 cells, 100 cells,90 cells, 80 cells, 70 cells, 60 cells, 50 cells, 40 cells, 30 cells, 20cells, 10 cells, 5 cells, 2 cells, or 1 cell. In some cases, a samplecontains at least about 10,000,000 cells, 5,000,000 cells, 1,000,000cells, 500,000 cells, 100,000 cells, 50,000 cells, 20,000 cells, 15,000cells, 10,000 cells, 9,000 cells, 8,000 cells, 7,000 cells, 6,000 cells,5,000 cells, 4,500 cells, 4,000 cells, 3,500 cells, 3,000 cells, 2,500cells, 2,000 cells, 1,500 cells, 1,000 cells, 900 cells, 800 cells, 700cells, 600 cells, 500 cells, 400 cells, 300 cells, 200 cells, or 100cells.

Samples of different masses can be processed on a fluidic device (e.g.,to extract and purify nucleic acids). For example, a sample can containfrom about 0.001 milligrams (mg) and about 10 mg of tissue. A sample cancontain at most about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg,0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg,0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg,5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of tissue. A sample can containat least about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg,0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg of tissue. A sample can containabout 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg,0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg,7 mg, 8 mg, 9 mg, or 10 mg of tissue.

Samples with different amounts of nucleic acid can be processed on afluidic device (e.g., to extract and purify nucleic acids). For example,samples can contain less than or equal to about 1 microgram (1 μg), 100nanograms (ng), 10 ng, 1 ng, 100 picograms (pg), 10 pg, or 1 pg ofnucleic acid. In some cases, samples can contain greater than or equalto about 1 microgram (1 μg), 100 nanograms (ng), 10 ng, 1 ng, 100picograms (pg), 10 pg, or 1 pg of nucleic acid.

Samples can be loaded in a sample buffer. The sample buffer may comprisea lysis agent or surfactant to lyse the input sample during off-chipprocessing to provide access to the target nucleic acids. The samplebuffer may comprise one or more leading electrolytes. The sample buffermay have sufficient wettability so as to self-load into the samplechannel due to gravity and/or surface tension. The sample buffer maycomprise one or more surfactants in order to reduce or minimizeadsorption of the target nucleic acids to the walls of the fluidicchannel. The sample buffer may comprise an ion content optimized to havesufficient salt for lysis and/or nucleic acid preservation while stillaccommodating the separation of nucleic acids. One of ordinary skill inthe art will understand that the higher the ion content of the samplebuffer (or any of the buffers described herein), the more current whichwill be required to transfer the charge of the sample buffer.

Samples can be loaded in a buffer comprising trailing electrolyte orleading electrolyte. Samples can be loaded in a buffer comprising asecond leading electrolyte which differs from the leading electrolyteused to perform ITP. Samples can be loaded in a buffer, such as anaqueous alkaline or a neutral aqueous buffer. Exemplary alkalinesolutions or buffers (e.g., for DNA extraction) can comprise 30-120 mMNaOH (in some cases, 40-80 mM NaOH) at a pH of about 10-13 (in somecases, with at least one additional component). In some instances, whenthe sample is lysed via treatment with an alkaline solution or bufferprior to loading onto the chip, the lysed sample may subsequently bequenched by adding an acidic solution or buffer to bring the pH of thelysed sample within a range of about 7.5 to about 8.5 prior toperforming isotachophoresis. Exemplary aqueous buffers (e.g., for DNA orRNA extraction) can comprise 2-150 mM Tris-HCl (at a pH of about 7 toabout 8) or BisTris-HCl at a pH of about 5.8 to about 7.3, with at leastone additional component. Additional components used in buffers caninclude non-ionic surfactants or detergents, ionic or zwitter-ionicsurfactants or detergents, chaotropic agents, disulfide bond reducingagents, proteases, nucleases, and other additives or components thatdigest, denature, disrupt, or degrade for the purpose of extracting,purifying, enriching, or otherwise isolating nucleic acids.

Samples can be loaded in a buffer comprising trailing electrolyte orleading electrolyte added to reduce or minimize retention of nucleicacids at a capillary barrier, particularly a capillary barrier between asample and LE. For example, a small amount of trailing electrolyte (e.g.MOPS and/or caproic) may be added to a lysate sample to intentionallyslow compaction of the DNA ITP band. Not wanting to be limited by aparticular theory, it is believed that this may help to maintain the DNAin a more dispersed state as it passes through a constricted space ofthe capillary barrier (e.g. a cliff capillary barrier at the junctionbetween the sample and LE) which may otherwise impair passage of a morecompact ITP band. Because the spiked-in TE has a slower magnitude ofmobility than both the DNA and the LE, once the ITP band enters the LEbuffer the TE may fall behind and allow the ITP band to fully compactbefore it reaches the elution reservoir.

Non-ionic surfactants or detergents can include but are not limited tosurfactants from the following classes: octylphenol ethoxylate,polysorbate, poloxamer, or polyoxyethylene. Octylphenol ethoxylatesurfactants can include but are not limited to branched octyiphenoxypolyethoxy ethanol (IGEPAL CA-630), t-octylphenoxypolyethoxyethanol(Triton™ X-100), or other polyethylene oxide chains with an aromatichydrocarbon lipophilic or hydrophobic group. Polysorbate surfactants caninclude but are not limited to polyethylene glycol sorbitan monolaurate(Tween 20), polyethylene glycol sorbitan monooleate (Tween® 80), orsorbitan monooleate (Span® 80). Poloxamer surfactants (i.e. blockcopolymers based on ethylene oxide and propylene oxide) can include butare not limited to polyoxyethylene-polyoxypropylene block copolymer(Pluronic® F-68) or polyethylene-polypropylene glycol block copolymer(Pluronic® F-127). Polyoxyethylene surfacts can include but are notlimited to nonyl phenoxypolyethoxylethanol (NP-40).

Non-ionic surfactants or detergents can include but are not limited toIGEPAL® (e.g., IGEPAL® CA-630), Triton™ X-100, Tween® 20, Tween® 80,NP-40, other block copolymers including Pluronic® (e.g., F-68 or F-127),Span® 80, and pegylated polymers or copolymers. Non-ionic surfactants ordetergents can be used to reduce or prevent biological moleculeadsorption to channel walls, or to control wetting and/or surfacetension properties of fluids to control loading of sample into fluidicdevices. Non-ionic surfactants or detergents can be present atconcentrations from about 0.0005-5% v/v or w/v. For example, IGEPALCA-630 can be used at about 0.05-0.5% v/v. Ionic surfactants ordetergents can include but are not limited to sodium dodecyl sulfate(e.g., at 0.01-2% w/v), sodium dodecylbenzenesulfonate (e.g., at 0.01-2%w/v), sodium cholesteryl sulfate (e.g., at 0.01%-2% w/v), and sodiumdeoxycholate (e.g., at about 10-1000 mM). Chaotropic agents can includebut are not limited to urea (e.g., at about 0.5-9.5 M, or in some cases,5-9.5 M) thiourea, butanol, ethanol, guanidinium chloride, lithiumperchlorate, lithium acetate, lithium chloride, magnesium chloride,phenol, and propanol. For example, 7.0 M urea and 2.0 M thiourea can beused in a 5-50 mM Tris-HCl (in some cases, 10-20 mM Tris-HCl) bufferedsolution for either RNA or DNA extractions, or for total nucleic acidextractions. The ratio of urea to thiourea can be at least about 1:1,1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 6.5:1, 7:1, 7.5:1,or 8:1. Disulfide bond reducing agents can include but are not limitedto DTT (e.g. at about 0.1-40 mM, or in some cases about 10 mM) andbetamercaptoethanol (e.g., at about 0.5-2%, or in some cases about 1%).Proteases can include but are not limited to Proteinase K, proteases,endoproteinases (e.g., trypsin, LysC, GluC, AspN), peptidases, pepsin,and papain. Nucleases can include but are not limited to non-specificnucleic acid digestion enzymes such as DNases including DNase I (e.g.,to prepare DNA-free RNA extractions) and RNase, such as RNase A, RNaseT, or combinations thereof (e.g., to prepare RNA-free DNA extractions).Nucleases can also include specific nucleic acid digestion enzymes(e.g., restriction enzymes) which can cut at specific nucleic acidsequences and can produce predictable fragment sizes and fragment sizedistributions. In some cases, one or more methods or processes providedherein are performed without use of a nuclease, without use of a DNAse,or without use of an RNAase. For example, the methods provided hereininclude extraction of RNA without use of DNAase.

Restriction enzymes can include but are not limited to Type 1 throughType V restriction enzymes, BamHI, EcoP15I, EcoRI, EcoRII, EcoRV,HaeIII, HgaI, HindIII, HinFI, KpnI, NotI, PstI, PvuII, SacI, SalI, SmaI,SpeI, SphI, XbaI, and StuI. Nucleases can be used at concentrationsincluding 50-400 μg/mL. Nuclease digestions can be performed attemperatures including from about 20° C. to about 37° C. Other nucleicacid modifying enzymes can be used, such as transposases, ligases,polymerases, and phosphatases. Other protein or polynucleotide digestionor degradation agents can be used, such as lysozymes.

Prior to loading onto a fluidic device, samples can be subjected tovarious degrees of pre-processing. In some cases, a sample can be simplyloaded into buffer prior to loading onto a fluidic device, and any othernecessary or desired sample preparation steps can be conducted on thedevice. In other cases, sample can be added to a sample reservoir thatis prefilled with a processing fluid such as a solution or buffer. Inother cases, a sample can be subjected to removal of embedding material,tissue disruption, cell lysis, or digestion prior to loading on afluidic device. In one example, a sample is deparaffinized prior toloading onto a fluidic device, and de-crosslinking of nucleic acids isconducted on the fluidic device. In another example, a sample isdeparaffinized, disrupted, and lysed prior to loading onto a fluiddevice, and, optionally, de-crosslinking of nucleic acids is conductedon the fluidic device. In another example, a sample is deparaffinizedprior to loading onto a fluidic device, and tissue disruption and celllysis are conducted on the fluidic device. In another example, a sampleis loaded onto a fluidic device, and deparaffinization, tissuedisruption, cell lysis, and de-crosslinking of nucleic acids are allconducted on the fluidic device. Sample preparation steps are discussedfurther in this disclosure.

Sample Preparation

Samples can be prepared prior to isotachophoresis. Sample preparationcan involve steps including but not limited to removal of embeddingmaterial, tissue disruption, cell lysis, digestion of proteins, removalof nucleic acid crosslinking, isothermal enzymatic process, enzymaticamplification, enzymatic digestion, disruption of cell-cell junctions,disruption of extracellular matrix, disruption of connective tissue, andcombinations thereof. Sample preparation can involve techniques such aspolymerase chain reaction (PCR) or other nucleic acid amplification,isolation or purification of material (e.g., cells, nucleic acids) ofinterest, probe hybridization, and antibody hybridization (e.g.,hybridization of antibodies to nucleosomes). In some cases, samples canbe prepared by isolating a portion of material from cells from thesample for further analysis. For example, circulating tumor cells can beisolated from a heterogenous population of cells using a cell sortingdevices such as a flow cytometer or magnetized column. In anotherexample, peripheral blood lymphocytes (PBLs) or peripheral bloodmononuclear cells (PBMCs) can be isolated from a blood sample. Samplepreparation can be conducted on-device or off-device. In some cases,some sample preparation steps are conducted off-device, and then thesample is loaded onto a fluidic device where additional samplepreparation steps are conducted.

Biological material (e.g., cells, tissue, nucleic acids) in an embeddedsample can be removed from the embedding material. For example, aparaffin-embedded sample can be deparaffinized. Removal of embeddingmaterial can be conducted using techniques including but not limited toheat treatment, chemical treatment (e.g., acid or base), enzymatictreatment, and combinations thereof. Deparaffinization can be performedby chemical treatment of a sample, by heat-treating a sample, byenzymatic treatment of a sample, or by other methods. For example,deparaffinization can be conducted at an elevated temperature (e.g. fromabout 50° C. to about 80° C.) in the presence of a neutral buffer orsomewhat acidic buffer (e.g., down to pH about 5.5) buffer or somewhatbasic (up to pH about 9) or alkaline solution (e.g., pH from about 12 toabout 13). Removal of embedding material can be conducted off-device oron-device. In one example, an embedded sample can be incubated at anelevated temperature in a vessel and subsequently loaded onto a fluidicdevice. In another example, an embedded sample can be loaded onto afluidic device and incubated at an elevated temperature on the device,for example in the channel or a reservoir.

Removal of embedding material can be conducted by heat treatment.Incubation for removal of embedding material can be conducted at atemperature of at least about 35° C., 37° C., 40° C., 45° C., 50° C.,55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C.,96° C., 97° C., 98° C., 99° C., 99.5° C., or 100° C. Incubation forremoval of embedding material can be conducted at a temperature fromabout 40° C. to about 80° C., from about 50° C. to about 80° C., fromabout 50° C. to about 99.9° C., or about 95 to about 99.5° C. Incubationfor removal of embedding material can be conducted for a duration of atleast about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes,110 minutes, 115 minutes, or 120 minutes. Incubation for removal ofembedding material can be conducted for a duration from about 1 minuteto about 20 minutes, from about 1 minute to about 30 minutes, from about1 minute to about 60 minutes, from about 1 minute to about 120 minutes,or from about 5 minutes to about 20 minutes. Incubation for removal ofembedding material can for example be conducted at a temperature of atleast about 37° C. for a duration of at least about 1 minute. Incubationfor removal of embedding material can be conducted in the presence of analkaline buffer or a neutral buffer (e.g. lysis buffer). An alkalinebuffer (e.g. lysis buffer) can have a pH of at least about 8.5, 9.0,9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, or 13.5. A neutral buffercan have a pH of about 7.0 (e.g., from about 7 to about 8).

Tissues or cells can be disrupted or lysed, releasing nucleic acids forseparation, purification, or extraction. Tissue disruption or cell lysiscan be conducted using techniques including but not limited tomechanical stress, sonication, electroporation, osmotic pressure,chemical treatment (e.g., acid or base), enzymatic treatment, heattreatment, and combinations thereof. For example, pressure can be usedto drive tissue through a structure (e.g., a channel, a resin such as afrit or porous resin, or a glass material) to mechanically disrupttissue or lyse cells. In some cases, the trailing electrolyte buffer cancomprise one or more tissue disruption agents and/or cell lysis agents.In some cases, the leading electrolyte buffer can comprise one or moretissue disruption agents and/or cell lysis agents. In some cases,removal of embedding material can be achieved by the same process astissue disruption or cell lysis. For example, incubation at an elevatedtemperature (e.g. from about 30° C. to about 80° C., from about 50° C.to about 80° C., or from about 30° C. to about 65° C.) can achieveremoval of embedding material, tissue disruption, and cell lysis. Tissuedisruption or cell lysis can be conducted off-device or on-device. Inone example, a tissue sample is disrupted in a vessel and subsequentlyloaded onto a fluidic device. In another example, a tissue samplepreviously loaded onto a fluidic device is disrupted on the device.

Samples comprising tissue or cells can be lysed before or after loadingonto a fluidic device using a lysis solution or buffer compatible withisotachophoresis. Lysis buffers compatible with isotachophoresis caninclude non-ionic surfactants or detergents, ionic or zwitter-ionicsurfactants or detergents, chaotropic agents, disulfide bond reducingagents, proteases, nucleases, and other additives or components thatdigest, denature, disrupt, or degrade for the purpose of extracting,purifying, enriching (concentrating), or otherwise isolating nucleicacids. In some cases, a lysis buffer may comprise an alkaline buffer. Insome cases, a lysis buffer may not comprise an alkaline buffer. Anexemplary lysis buffer may include 0.5 M to 9.5 M, 4 M to 9 M, or 6.5 Mto 7 M urea as described herein. An exemplary lysis buffer may include0.5 M to 3.5 M or 1.5 M to 2.5 M thiourea as described herein. Anexemplary lysis buffer may include 0.5-9.5 M urea and thiourea, forexample 7M urea and 2 M thiourea with a non-ionic surfactant asdescribed herein. The use of urea alone or in combination with thioureamay be used to lyse cells for nucleic acid purification. In combination,urea and thiourea may act synergistically to lyse cells and may providean uncharged isotachophoresis-compatible buffer for nucleic acidpurification.

An exemplary lysis buffer may include a non-ionic surfactant such as0.05-0.5% v/v IGEPAL CA-630 as described herein. In some cases, thelysis buffer may comprise one or more trailing electrolytes. In somecases, the lysis buffer may comprise a trailing electrolyte buffer withadditives for tissue disruption or cell lysis as described herein. Insome cases, the lysis buffer may comprise one or more leadingelectrolytes. In some cases, the lysis buffer may comprise a leadingelectrolyte buffer with additives for tissue disruption or cell lysis asdescribed herein. In some cases, the lysis buffer may comprise one ormore leading electrolytes and one or more trailing electrolytes. In somecases, the lysis buffer may comprise one or more leading electrolytesand one or more trailing electrolytes with additives for tissuedisruption or cell lysis as described herein.

In some cases, a method or process herein may involve lysing a cell ortissue sample using a lysis buffer that minimizes mechanical disruptionof DNA and/or RNA during the lysis reaction. For example, cells ortissue may be lysed in a buffer solution containing Tris (e.g., 5 mM, 10mM, 20 mM, 30 mM Tris) with HCl (e.g., 1 mM, 5 mM, 10 mM HCl) and anon-ionic surfactant. The non-ionic detergent (e.g., IGEPAL CA-630) maybe present at about 1%, about 2%, about 3%, about 4%, or greater in thelysis buffer, or less than about 1%. Cells or tissue may be lysed in thelysis buffer by gentle mixing such as by inversion and low-speed(automated pipette). An enzyme such as proteinase K may, in some cases,be included in the lysate or lysis buffer. In some cases, the lysis isconducted without centrifugation. In some cases, centrifugation is usedin the lysis method. The lysate may be introduced into anisotachophoresis device in order to purify a desired analyte such ashigh molecular weight DNA fragments.

Proteins in a sample can be digested, for example via enzymaticdigestion with proteases. Proteases can include but are not limited toProteinase K, proteases, endoproteinases (e.g., trypsin, LysC, GluC,AspN), peptidases, pepsin, and papain. Other protein or polynucleotidedigestion or degradation agents can be used, such as lysozymes.Digestion of proteins can remove crosslinking proteins from crosslinkednucleic acids, converting them into non-crosslinked nucleic acids.Digestion of proteins can occur at room temperature or at elevatedtemperatures described herein (e.g. greater than about 25° C.).

Sample can be processed on a device (e.g., an electrokinetic device orsystem with at least one reservoir connected to at least one channel),such that the sample volume passes through the reservoir into thechannel with less than 20% of the sample volume left behind in thereservoir, and subsequently an ionic current can be applied through thesample volume in the channel. The ionic current may not substantiallypass through the channel. In some cases, less than 50%, 45%, 40%, 35%,30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, or 1% of the sample volume is left behind in thereservoir.

Sample can be processed on a device (e.g., an electrokinetic device orsystem with at least one reservoir connected to at least one channel),such that the sample volume which passes through the reservoir into thechannel is at least 50% of the sample volume loaded into the reservoir,and subsequently an ionic current can be applied through the samplevolume in the channel. In some cases, at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99%, or more of the sample volume is moved fromthe reservoir to the channel. In some instances, the total volume loadedinto the reservoir is less than or equal to an internal volume of thereservoir. The ionic current may not substantially pass through thechannel. In some cases, applying an ionic current comprises conductingisotachophoresis.

In some embodiments, after the sample volume has been loaded into thereservoir, and subsequently loaded into the channel, a topper buffer maybe added to the reservoir to facilitate movement of the sample volumeinto the channel. A volume of topper buffer may be added to thereservoir to “push” additional sample volume into the channel. Forexample, a volume of topper buffer greater than or equal to the volumeof sample left behind in the reservoir may be added to the reservoir soas to move at least a portion of the remaining sample volume into thechannel. The topper buffer may, for example, comprise the same buffer asthe sample buffer but have no analyte therein.

Isotachophoresis Devices

Isotachophoresis and/or sample preparation (e.g., deparaffinization,digestion, lysis) can be conducted in a fluidic device, for example amicrofluidic chip. For example, FIG. 5A shows a schematic of a channelwith a sample preparation (e.g., deparaffinization) zone 500 with asample inlet 501 and a trailing electrolyte reservoir 502, apurification (e.g., isotachophoresis) zone 510 with a leadingelectrolyte reservoir 511, and an elution outlet 520. A capillarybarrier may provide an interface between the sample fluid and theleading electrolyte buffer prior to applying voltage. A capillarybarrier may be provided between the sample preparation zone 500 and thetrailing electrolyte reservoir 502 in order to limit, reduce, or preventmixing or pressure-driven flow of the sample fluid and the trailingelectrolyte buffer. A capillary barrier may be provided between thepurification zone 510 and the leading electrolyte reservoir 511 so as tolimit, reduce, or prevent mixing or pressure-driven flow of the contentsof zone 510 and the leading electrolyte reservoir 511. In anotherexample, deparaffinization can be performed first off-chip, or can beunnecessary due to the starting material, in which case the channel cancomprise a lysis and digestion zone (e.g., pH 7, 56° C.) and a crosslinkremoval and purification (e.g., isotachophoresis) zone (e.g., pH 7, 80°C.). In another example, deparaffinization can be performed firstoff-chip, or can be unnecessary due to the starting material, in whichcase the channel can comprise a lysis and digestion zone (e.g., pH 7,temperature T1) and a crosslink removal and/or purification (e.g.,isotachophoresis) zone (e.g., pH 7, temperature T2). In another example,deparaffinization can be performed first off-chip, or can be unnecessarydue to the starting material, in which case the channel can comprise adisruption and/or lysis zone (e.g., pH 7, temperature T1) and adigestion and/or purification (e.g., isotachophoresis) zone (e.g., pH 7,temperature T2). In another example, deparaffinization can be performedfirst off-chip, or can be unnecessary due to the starting material, inwhich case the channel can comprise a disruption and/or lysis zone(e.g., pH 7, temperature T1) and an isothermal enzymatic amplificationzone (e.g., pH 7, temperature T2). In another example, deparaffinizationcan be performed first off-chip, or can be unnecessary due to thestarting material, in which case the channel can comprise a disruptionand/or lysis zone (e.g., pH 7, temperature T1) and an isothermalenzymatic digestion zone (e.g., pH 7, temperature T2). In some cases,the channel may comprise three zones, for example a disruption and/orlysis zone (e.g. pH 7, temperature T1), an isothermal enzymaticamplification zone (e.g., pH 7, temperature T2), and a purification(e.g. isotachophoresis) zone (e.g. pH 7, temperature T3). FIG. 5B showsan exemplary fluidic device cartridge with eight parallel channels eachas shown in FIG. 5A. FIG. 5C shows a top-view schematic of the fluidicdevice shown in FIG. 5B, while FIG. 5D and FIG. 5E show side and endviews, respectively. The devices can comprise sample inlets orreservoirs 530, ITP electrolyte buffer reservoirs 531, and sampleelution outlets or reservoirs 532. The channels and/or reservoirs may becoupled to one or more pneumatic ports. Each of the eight parallelchannels of the fluidic device may be independently operated from eachof the other channels. In some cases, each channel has a dedicated setof electrodes and electric circuitry to drive ITP. Electrodes may forexample be located in the trailing electrolyte reservoir 502 and theleading electrolyte reservoir 511 such that the electrodes do notdirectly contact sample material.

In some instances, there may be little or no fluid or ion flow betweenparallel channels. In some cases, the parallel channels may not be influid communication with one another. The fluid leakage rate betweenparallel channels may be less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1 μL per hour.

In some instances, there may be little or no electrical communicationbetween parallel channels such that the parallel channels areelectrically isolated from one another. Each of the parallel channelsmay be independently electrically controlled so as to apply anindependent electric field to each of the channels. In some instances,current leakage between the channels is less than about 0.1 microamperes(μA), 0.2 μA, 0.3 μA, 0.4 μA, 0.5 μA, 0.6 μA, 0.7 μA, 0.8 μA, 0.9 μA, or1 μA. In some instances, the impedance between channels may be greaterthan 0.1 mega Ohm (MOhm), 0.2 MOhm, 0.3 MOhm, 0.4 MOhm, 0.5 MOhm, 0.6MOhm, 0.7 MOhm, 0.8 MOhm, 0.9 MOhm, 1 MOhm, 5 MOhm, 10 MOhm, 20 MOhm, 30MOhm, 40 MOhm, or 50 MOhm.

In some instances, each of the parallel channels may be coupled to thesame current or voltage source and independently electricallycontrolled. In some instances, each of the parallel channels may becoupled to a different current or voltage source and independentlyelectrically controlled.

FIG. 34 shows a circuit configured to detect and prevent current leakageduring ITP in parallel channels. The circuit may be configured tomonitor the current into the ITP system from a single HV electrode andthe current that leaks through the high-voltage board. By measuring thetwo currents at regular intervals or continuously, the current into thechip can be precisely controlled even if the leakage current changes.This may allow the electrode to be run in either net source or net sinkconfiguration. The circuit may comprise a voltage source Vs, a sinkcurrent controller 3406, a source current controller 3408, and twocurrent measurement circuits 3405 and 3409 that measure the currentflowing through the controlled current sources 3406 and 3408. Thecontrolled current sources can be controlled by a microprocessor throughthe use of digital-to-analog (DAC) convertors 3402 and 3403. DACconverter 3402 may control current into the electrode and the leak path.DAC converter 3403 may control current into the leak path. The measuredcurrents can be read by a microprocessor through the use ofanalog-to-digital converters (ADC) 3401 and 3404. ADC 3401 may measurecurrent at the source Vs. ADC 3404 may measure current at the leak path,which is directed to ground. The current controlled sources 3406 and3408 may be joined with a load 3407 so that the current source 3408 cansource a current into the load 3407 and so that the current source 3406can sink a current from the load 3407. The current that flows into theload 3407 can be denoted I_(RL). The current that flows through thecontrolled current source 3408 can be denoted I_(m1). The current thatflows through the controlled current source 3406 can be denoted I_(m2).The current that will flow in to or out of the load 3407 can thus bedescribed by the equation I_(RL)=I_(m1)−I₂. The current I_(m1) iscomprised of both the current commanded by the controlled source 3408which will be denoted by I_(c1) and a leakage current denoted by I_(L1)due to parasitic conductive paths present in any physically realizablecircuit. The current I_(m1) can be described by the equationI_(m1)=I_(c1)+I_(L1) and similarly the current I_(m2) can be describedby I_(m2)=I_(c2)+I_(L2). By extension, the current flowing in to or outof load 3407 can thus be described by the equationI_(RL)=I_(c1)+I_(L1)−I_(c2)−I_(L2). In some applications it may bedesired to command the circuit to an off-state so that I_(RL)=0. Toaccomplish this, the controlled current sources will typically becommanded so that I_(c1)=0 and I_(c2)=0, however, due to the leakagecurrents I_(L1) and I_(L2), the current flowing in to or out of the load3407 during the off-state may be some non-zero current described byI_(RL)=I_(L1)−I_(L2). In order to reduce the off-state current I_(RL) toa value significantly less than the leakage currents I_(L1) or I_(L2),the circuit can steer additional current through either the controlledcurrent source 3406 or 3408 until the balance of current flowing in toI_(RL) is nulled. For example, the circuit can set eitherI_(c1)=I_(c2)+I_(L2)−I_(L1) or I_(c2)=I_(c1)+I_(L1)−I_(L2). In eithercase, the resulting current I_(RL) will reduce to I_(RL)=0. In otherwords, the circuit may adjust current(s) from the current source(s)3406, 3408 to balance and counteract the leakage current to drive thenet flow between parallel channels to 0.

In many applications where a controlled current source is to be appliedto a load for a period of time and then removed from the load for aperiod of time, such as in isotachophoresis, it may be desirable for thecurrent source circuit to leak as little current into the load when thecontrolled current source is to be removed. Many or all circuitcomponents used to construct current sources may allow for someparasitic leakage current to flow through the circuit when the controlcircuit is intended to be off. Minimizing this leakage typicallyrequires the use of more sophisticated and higher quality componentsthat exhibit lower parasitic leakage properties, however, doing so comesat higher cost and often requires more physical volume to implement thecircuit. The disclosure disclosed allows for the leakage current appliedto a load to be reduced by steering the leakage current away from theload. The disclosure thus may allow for simpler circuit components to beused in the construction of the current source enabling a current sourcethat may realize a lower leakage current with circuit components thatare optimized for other purposes such as lower cost or physical size.Current leakage may result from liquid leaking between fluidic channelswhere a layer of material closes fluidic channels in a surface of asubstrate. Ensuring secure bonding of the layer across the substratesurface may reduce such leakage. Current leakage also can result fromliquid moving between ports of different fluidic circuits, in particularports that are a source of negative pressure to a fluidic channel.Provision of hydrophobic barriers at such ports may reduce such leakage.

In some instances, each zone on the isotachophoresis device can beheated. In some instances, the zones are heated to the same temperature.In some instances, individual zones are heated to differenttemperatures. In some instances, a first zone may be heated to atemperature above 37° C., for example within a range of about 60° C. toabout 100° C. In some instances a second zone may be heated to atemperature above 37° C., for example within a range of about 40° C. toabout 60° C.

An isotachophoresis fluidic device can comprise one or more reservoirs,including but not limited to buffer loading reservoirs, sample loadingreservoirs (including reservoirs that accept solid, multiphasic, orother inhomogeneous liquids or solutions such as tissue, whole blood, orunlysed cell suspensions), leading electrolyte reservoirs, trailingelectrolyte reservoirs, reagent reservoirs, elution reservoirs (e.g.,for unloading processed samples), and gas or air reservoirs. In somecases, one physical reservoir can be used for multiple purposes, such asbuffer loading and sample loading. Liquid or air reservoirs can be usedto apply external pressure for liquid loading (e.g., positive pressureon liquid wells or vacuum on gas only reservoirs). It will be understoodby one of ordinary skill in the art that any of the reservoirs describedherein may be used to load or retrieve any of the buffers and/or samplesdescribed herein.

Reservoirs can be in thermal communication with a heating or coolingsource, allowing control of the temperature of the reservoir and anymaterial within (e.g., reagent, sample, product). For example, anelution reservoir can be thermally controlled to control the temperatureof the eluted product (e.g., for preservation of structure, integrity)while within the fluidic device.

Reagent reservoirs can be used to load one or more reagents forprocessing the sample before, during, or after isotachophoresis.Reagents can include digestion reagents, amplification reagents, reversetranscription reagents, linear polymer solutions for size-basedseparations, probes for hybridization reactions, ligation reagents, dyes(e.g. intercalating dyes described herein), tracers, labels, and otherreagents. Reagent reservoirs can be connected to a reaction channel, ora reaction section of another channel, where reactions can occur.Heating or cooling can be applied (e.g., with thermal controllers asdiscussed herein) to catalyze reactions (such as enzymatic reactionswith nucleic acids or proteins), to hybridize or melt nucleic acids, orremove intercalated dyes from nucleic acids (for example, prior toelution). Heating and cooling can also be used to control a fixedoperating temperature for conducting ITP (e.g., cooling can be appliedto reduce effects of Joule heating), or to keep a reservoir (e.g., anelution reservoir) at a fixed temperature (e.g., cooler than roomtemperature), such as for stable storage of purified nucleic acids.Light can be applied (e.g., with light sources as discussed herein) forpurposes including optical interrogation, fluorescent excitation, andreaction energy or catalysis.

Gas or air reservoirs, or gas or air outlets, can be connected via gaschannels to liquid channels within a fluidic device to allow purging ofair or other gases from the fluidic device (e.g., during liquid fillingof the fluidic device). Gas or air reservoirs, or pneumatic pressureports, can be connected via gas channels to liquid channels to allow forpumping of fluids onto or within the fluidic device (e.g., for pumpingof fluids from reservoirs into channels).

A device can comprise multiple purification (e.g., isotachophoresis)zones in connection with each other. For example, a secondisotachophoresis zone can split from and run in parallel to a firstisotachophoresis zone, allowing splitting of a sample band at aspecified ratio (e.g., based on a ratio of currents between the twozones) for parallel processing.

A fluidic device can comprise multiple purification zones in parallel(see, e.g., FIG. 5C). For example, a fluidic device can comprise morethan one set of purification zones, each with associated reservoirs,inlets, outlets, channels, and any other components described herein(e.g., sample preparation zones, electrodes, heaters, detectors) inparallel, separate from each other and each capable of independentlyprocessing a sample. A fluidic device can comprise at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 48, 96, ormore purification zones in parallel. A fluidic device can comprisemultiple channels in parallel. A fluidic device can comprise at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 48,96, or more channels in parallel. Components, such as purification zonesor channels, located in parallel can be side-by-side, located indifferent device layers (e.g., horizontal or vertical layers), or placedin different arrangements. Parallel components can be identical, or canbe designed differently but function equivalently or nearlyequivalently. For example, parallel channels can have differentgeometries to allow a smaller overall fluidic device footprint, butstill function similarly. Alternatively, parallel components can bedesigned to function differently, for example to process different typesof samples in parallel, or to subject samples to different operations.In some cases, parallel components can be designed to subject differentsample types to different operations in parallel. In some cases,parallel components can be designed to subject the same sample types todifferent operations in parallel. In some cases, parallel components canbe designed to subject different sample types to the same operations inparallel. In some cases, parallel components can be designed to subjectthe same sample types to the same operations in parallel. In some cases,parallel components can be designed to simultaneously and/orindependently subject two or more samples to one or more operations inparallel. In some cases, a leakage rate between two or more channels (orbetween two or more purification zones) may be less than 0.5 μper hour,less than 1 μl per hour, less than 5 μl per hour, less than 10 μl perhour. In some embodiments, a current leakage rate between two or morechannels (or between two or more purification zones) may be less than0.5 μA, less than 1 μA, less than 5 μA, or less than 10 μA. In someembodiments, an impedance between channels or zones may be greater than0.5 megaOhm, greater than 1 megaOhm, greater than 5 megaOhm, or greaterthan 10 megaOhm.

As discussed herein, a fluidic device can be designed to processdifferent sample volumes. For example, FIG. 6A, FIG. 6B, FIG. 6C, andFIG. 6D show top, side, bottom, and top three-quarters views,respectively, of a rapid purification ITP fluidic device 600 for samplevolumes greater than or equal to about 200 μL. The device comprises achannel 600 connected to sample input wells 601, ITP buffer wells 602,and sample output (elution) wells 603 by through-holes or apertures asdescribed herein. The ITP buffer wells 602 can include an elutionbuffering reservoir 605, a leading electrolyte reservoir 606, a leadingelectrolyte buffering reservoir 607, and a trailing electrolytereservoir 608. Elution reservoir 603 may be connected to elutionbuffering reservoir 605 by an elution buffering channel 609. A capillarybarrier (e.g. a plateau capillary barrier, a ramp capillary barrier, ora cliff capillary barrier as described herein) may be provided in theelution buffering channel 609 to reduce or prevent mixing or pressuredriven flow between the contents of the elution buffering reservoir 605and the elution reservoir 603. Leading electrolyte reservoir 606 may beconnected to leading electrolyte buffering reservoir 607 by a leadingelectrolyte buffering channel 610. A capillary barrier (e.g. a plateaucapillary barrier, a ramp capillary barrier, or a cliff capillarybarrier) may be provided in the leading electrolyte buffering channel610 to reduce or prevent mixing or pressure-driven flow between thecontents of the leading electrolyte buffering reservoir 607 and theleading electrolyte reservoir 606. Buffering reservoir 605 may containelution buffer electrolytes at a higher ionic strength than those inelution reservoir 603, while buffering reservoir 607 may contain leadingelectrolytes at a higher ionic strength than those in leadingelectrolyte reservoir 606. The device may further comprise pneumaticports 604 along its edges which are configured to couple to a pneumaticdevice, for example a vacuum source on a benchtop instrument. Thepneumatic ports 604 may be coupled to the channel 600 and reservoirs bygas channels as described herein. Application of suction at thepneumatic ports 604 may load the sample, leading electrolyte, andelution buffer into the channel 600. In some cases, the trailingelectrolyte buffer fluid remains in the trailing electrolyte reservoir608. Suction may be applied simultaneously or sequentially to thepneumatic ports 604 so as to load the channel 600 simultaneously or instages, respectively. The sample may be loaded into a first zone orsub-channel of channel 600 which extends from the trailing electrolytereservoir 608 to a capillary barrier 611 at a 180° low dispersion turnin the channel 600. The capillary barrier 611 may provide an interfacebetween the sample and the leading electrolyte buffer during loading soas to limit, reduce, or prevent mixing or pressure-driven flow. Thecapillary barrier 611 may comprise a cliff capillary barrier asdescribed herein. A capillary barrier (e.g. a cliff capillary barrier, aramp capillary barrier, or a plateau capillary barrier) may be providedbetween the trailing electrolyte reservoir 608 and the first zone orsub-channel so as to limit, reduce, or prevent mixing or pressure-drivenflow between the contents of the trailing electrolyte reservoir 608 andthe sample. The leading electrolyte may be loaded into the second zoneor sub-channel of the channel 600 which extends from capillary barrier611 to capillary barrier 612. The capillary barrier 612 (e.g. a plateaucapillary barrier, a ramp capillary barrier, or a cliff capillarybarrier) may provide an interface between the leading electrolyte bufferand the elution buffer. The elution buffer may be loaded into a thirdzone or sub-channel of channel 600 which extends from capillary barrier612 to elution reservoir 603. In some embodiments, the ITP buffer wells602 may further comprise a trailing electrolyte buffering reservoir (notshown) containing trailing electrolytes at a higher ionic strength thanthose in the trailing electrolyte reservoir 608. The trailingelectrolyte buffering reservoir may be connected to the trailingelectrolyte reservoir 608 by a trailing electrolyte buffering channel(not shown). The trailing electrolyte buffering channel may comprise acapillary barrier (e.g. a ramp capillary barrier, a plateau capillarybarrier, or a cliff capillary barrier) to limit, reduce, or preventmixing or pressure-driven flow between the contents of the trailingelectrolyte buffering reservoir and the trailing electrolyte reservoir608.

Electrodes may for example be located in the trailing electrolytereservoir 608, a trailing electrolyte buffering reservoir (not shown),the leading electrolyte reservoir 606, and/or the leading electrolytebuffering reservoir 607 such that the electrodes do not directly contactsample material. The electrodes may be triggered to alter or control theapplied electric field in response to feedback from a sensor, forexample a voltage, current, conductivity, or temperature sensor asdescribed herein. For example, passage of the nucleic acids within theITP zone from the second zone of channel 600 to the third zone ofchannel 600 may be detected and feedback from the detector may triggerthe applied current to change. The current may for example be increased,decreased, or ended according to the protocol of the instrument. Thecurrent may for example be paused (e.g. dropped temporarily to zero) inorder to enable on-chip quantification of the nucleic acids.Alternatively or in combination, the current may be decreased in orderto slow isotachophoresis within the third zone to allow the nucleicacids which may have dispersed upon transition from the leadingelectrolyte buffer to the elution buffer (or second leading electrolytebuffer) time to concentrate further before reaching the elution well603.

The methods and processes provided herein include methods and processesthat use any of the devices provided herein. Devices provided hereinwith multiple channels for processing multiple samples in parallel maybe used in a variety of contexts. In some cases, a method may includeuse of a device to process multiple samples (e.g., by conductingisotachophoresis on such samples) that share a certain feature (e.g.,solid tissue lysate, cell lysate, solid tissue, fixed tissue). In somecases, the multiple samples may be different samples. For example, themethod may involve performing isotachophoresis on a tissue sample in onezone of the device while simultaneously, but independently, conductingisotachophoresis on a different sample such as a cellular sample orsample comprising cross-linked nucleic acids.

In some cases, a method or multiplexing process provided herein mayinvolve conducting isotachophoresis on a sample in a channel in parallelwith conducting isotachophoresis on a second sample in a second channelusing leading electrolyte and/or trailing electrolyte buffers that arethe same or similar. In some cases, a sample in one of the channels isprocessed using a first leading electrolyte buffer and a sample in adifferent channel is processed using a second leading electrolyte bufferthat is different from the first. For example, the first leadingelectrolyte buffer can contain one or more leading electrolyte ions thatare different from those contained in the second leading electrolytebuffer. In another example, the first leading electrolyte buffer cancontain one or more leading electrolyte ions that are the same as thosecontained in the second leading electrolyte buffer but the concentrationof such leading electrolyte ions in the first leading electrolyte bufferis different from the concentration of such ions in the second leadingelectrolyte buffer. In some cases, a method or process provided hereinmay involve conducting isotachophoresis on a sample in a channel inparallel with conducting isotachophoresis on a second sample in a secondchannel using trailing electrolyte or trailing electrolyte buffers thatare the same or similar. In some cases, a sample in one of the channelsis processed using a first trailing electrolyte buffer and a sample in adifferent channel is processed using a second trailing electrolytebuffer that is different from the first. For example, the first trailingelectrolyte buffer can contain one or more trailing electrolyte ionsthat are different from those contained in the second trailingelectrolyte buffer. In another example, the first trailing electrolytebuffer can contain one or more trailing electrolyte ions that are thesame as those contained in the second trailing electrolyte buffer theconcentration of such trailing electrolyte ions is different in thefirst trailing electrolyte buffer is different from the concentration inthe second trailing electrolyte buffer.

In some embodiments, one or more reservoirs may be connected to twochannels or sub-channels. For example, elution reservoir 603 may beconnected to both channel 600 and elution buffering channel 609.Alternatively or in combination, leading electrolyte reservoir 606 maybe connected to both channel 600 and leading electrolyte bufferingchannel 610. Alternatively or in combination, trailing electrolytereservoir 608 may be connected to both 600 and a trailing electrolytebuffering channel. Alternatively or in combination, sample input well601 may be connected to a mid-point in channel 600 such that channel 600extends to the left (as a first sub-channel) and right (as a secondsub-channel) of the input well 601. The two channels or sub-channels maybe connected to the one or more reservoirs with an angle between the twochannels (swept in the major plane of the fluidic device) of at leastabout 5°, 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°, 80°, 90°, 100°, 110°,120°, 130°, 135°, 140°, 150°, 160°, 170°, or 180°. The two channels orsub-channels may be connected to the one or more reservoirs with anangle between the two channels (swept in the major plane of the fluidicdevice) of at most about 5°, 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°,80°, 90°, 100°, 110°, 120°, 130°, 135°, 140°, 150°, 160°, 170°, or 180.

The device may comprise, for example, 8 channels as shown. Each channelmay hold a sample volume of about 50 μL to about 275 μL and a totalvolume of about 500 μL. The 180° low dispersion turn in each channel mayfacilitate such large sample volumes in an 8-channel multi-channel platewith a standard SLAS footprint.

FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D show top, side, bottom, andbottom three-quarters views, respectively, of a rapid purification ITPfluidic device 700 for sample volumes less than or equal to about 100μL. The device comprises sample input wells 701, ITP buffer wells 702,and sample output (elution) wells 703. The device 700 may besubstantially similar to device 600 but with different channel geometry(and corresponding reservoir geometry) that does not include a 180° turnin the channel.

The device may comprise, for example, 8 channels as shown. Each channelmay hold a sample volume of about 10 μL to about 100 μL. A device withsmaller sample volumes may be useful for PCR cleanup or other reactioncleanup applications or for smaller sample sizes (for example a samplewith a low number of cells or a small amount of tissue).

FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D show top, side, bottom, andthree-quarters bottom views, respectively, of another rapid purificationITP fluidic device 800 for sample volumes less than or equal to about100 μL. The device comprises sample input wells 801, ITP buffer wells802, and sample output (elution) wells 803. The device 800 may besubstantially similar to devices 600 and 700 but comprises multipledifferent channel geometries on a single chip.

Any of the fluidic devices described herein can comprise one or moreelectrodes that apply an electric field to a fluidic device or a part ofthe fluidic device. Applied electric fields can be used for conductingisotachophoresis. The fluidic device may comprise one or more electrodesthat apply a single electric field to all channels of the fluidicdevice. The fluidic device may comprise one or more electrodes thatapply more than one electric field to the fluidic device, for exampleone electric field per channel on the device. In some instance, a firstand second electric field are generated from a single electrode pair. Insome instances, a first and second electric field are generated fromdifferent electrode pairs. The electric fields may be appliedsimultaneously, sequentially, and/or independently or one another.Electrodes can be external, such as a wire that drops into a reservoir.Electrodes can be internal, such as a microfabricated, printed, or otherembedded element included within the fabrication of the fluidic device.Electrode materials can include but are not limited to metals (e.g.,platinum, titanium), and carbon.

The one or more electrodes of the fluidic device may be part of one ormore electric circuits that apply an electric field to a fluidic deviceor part of a fluidic device. The fluidic device may comprise one or moreelectric circuits that apply a single electric field to all channels orisotachophoresis regions of zones of the fluidic device. The fluidicdevice may comprise one or more electric circuits that apply more thanone electric field to the fluidic device, for example one electric fieldper channel on the device. In some instance, first and second electricfields may be generated from a single electric circuit. In someinstances, first and second electric fields may be generated fromdifferent electric circuits. The electric fields may be appliedsimultaneously, sequentially, and/or independently or one another by theone or more electric circuits. In some instances the device (or benchtopinstrument) may be configured to control a first electric circuitsimultaneously with and independently of a second electric circuit.

Electrodes can be located in reservoirs, such as trailing and leadingelectrolyte reservoirs, which can be separated from sample reservoirs bybuffering channels. In some cases, electrodes are located in bufferingchannels or buffering reservoirs. Location of electrodes in electrolytereservoirs or electrolyte buffering reservoirs can isolate theelectrodes from analytes such as nucleic acids to reduce or eliminatecontamination of electrodes by sample material. This approach can allowreuse of electrodes without cross-contamination between samples. In oneexample, a trailing electrolyte reservoir or trailing electrolytechannel is connected by a buffering channel to a buffering reservoirwhich contains trailing electrolyte ions and an electrode, and thetrailing electrolyte reservoir is also connected to a sample reservoiror sample channel, which in turn is connected to a leading electrolytereservoir by a leading electrolyte channel; the leading electrolytereservoir is also connected by a buffering channel to a bufferingreservoir which also contains leading electrolytes and an electrode. Inanother example, or as a continuation of the previous example, anelution reservoir containing elution buffer is connected to a leadingelectrolyte reservoir by an elution channel and is also connected to abuffering reservoir containing elution buffer electrolytes and anelectrode. The buffering channels between the buffering reservoirs andtheir corresponding reservoirs can include capillary barriers and/or alow cross-sectional area to limit, reduce, or prevent mixing andpressure-driven flow as described herein. The buffering reservoirs maycontain electrolytes at the same or higher ionic strength as theircorresponding reservoirs. For example, the elution reservoir can beconnected to a buffering reservoir containing elution bufferelectrolytes at the same or higher ionic strength or concentration asthe elution reservoir. The trailing electrolyte reservoir can beconnected to a buffering reservoir containing trailing electrolytes atthe same or higher ionic strength or concentration as the trailingelectrolyte reservoir. The leading electrolyte reservoir can beconnected to a buffering reservoir containing leading electrolytes atthe same or higher ionic strength or concentration as the leadingelectrolyte reservoir. Providing dedicated buffering reservoirsconnected to the elution reservoir, trailing electrolyte reservoir,and/or leading electrolyte reservoir with higher ionic strengths canprovide a pool of additional ions to maintain pH and conductivity in thechannel as the sample moves through the channel.

Fluidic devices can be used with one or more thermal controllers. Forexample, FIG. 9A shows a schematic of an eight-plex sample preparationand isotachophoresis device, comprising eight parallel channels 900 ofthe design shown in FIG. 5A. FIG. 9B shows a schematic of a first and asecond thermal controller 901, 902. A first thermal controller 901 attemperature T1 (e.g., 80° C.) is aligned with the sample preparationzones of the channels and a second thermal controller 902 at temperatureT2 (e.g., 50° C.) is aligned with the isotachophoresis zones of thechannels. In some cases, additional thermal controllers may be alignedwith additional zones of the channels (not shown), for example a thirdthermal controller at temperature T3 may be aligned with a third zone attemperature T3. In some cases, each zone of each channel can have itsown separate thermal controller, rather than sharing a common thermalcontroller with the respective zones of the other channels. In othercases, all the zones or channels can share one thermal controller. Inother cases, more than one but less than all the zones or channels canshare one thermal controller. Thermal controllers can comprisecomponents including but not limited to resistive heaters, fluid-basedheating or cooling systems, and Peltier devices. Thermal controllers canbe fabricated from materials including but not limited to metals (e.g.,platinum, titanium, copper, gold), carbon, and indium tin oxide (ITO).Thermal controllers can comprise temperature sensors, which can be usedto monitor the temperature being controlled and provide temperaturefeedback for thermal control. Thermal controllers can be used withcomputer control systems, as discussed further in this disclosure. Insome cases, thermal controllers are operated without temperaturefeedback. Thermal controllers can be integrated into fluidic devices orlocated externally, such as within a benchtop system.

Fluidic devices can be used with one or more light sources. Lightsources can be integrated into fluidic devices or located externally toa fluidic device, such as within a benchtop system or in a separatedevice. Light sources can provide light for optical interrogation,fluorescent excitation, temperature sensing, reaction energy orcatalysis, and other purposes.

Fluidic devices can be designed such that their outermost frame ordimensions meet microtiter plate standards (e.g., SLAS microtiter platestandards). Fluidic devices can be designed to use the defined ports ofa microtiter plate (e.g., SLAS standard microtiter plate) as liquidreservoirs, with pneumatic actuation ports located on the unused surfaceexternal to the liquid reservoirs. Pneumatic ports can be arranged atthe edges of a fluidic device with a microtiter plate-compatible layoutsuch that cross-contamination through pneumatic actuation across liquidreservoirs is avoided, and such that the ports are easy to access withpneumatic hardware. A subset of defined ports can also be used forpneumatic actuation in addition to their other functions. In some cases,a fluidic device can be designed and fabricated in two interlockingparts: first, an insert that includes a channel unit (e.g. a layer witha flat surface enabling ease of film bonding), wells, and pneumaticports; and second, an outer ring or cover piece to provide conformity toa microtiter plate standard (e.g., SLAS microtiter plate dimensionalstandards), including alignment features for aligning the fluidic deviceto a benchtop system and mating features to interlock with the firstpart. Wells can be connected to form bosses, which can be morecompatible with injection molding. In some cases, a fluidic device canbe designed and fabricated in three connecting parts; first, a chip orsubstrate that includes wells and pneumatic ports on its top face andetched or molded channels on its bottom face, second, a layer ofmaterial (e.g. a film) which seals to the bottom face of the chip toform closed channels (which, together, suffice to form a fluidic chip)and, third, a cover piece an outer ring or cover piece to provideconformity to a microtiter plate standard (e.g., SLAS microtiter platedimensional standards), including alignment features for aligning thefluidic device to a benchtop system and mating features to interlockwith the first part. Such a device can also be referred to as acartridge.

FIGS. 35A-35B show an example of a fluidic device 3500 which comprisestwo interlocking parts. FIG. 35A shows a cover piece 3501 which fitsonto a microfluidic chip insert part 3502 as shown in FIG. 35B. FIG. 35Bshows an exploded view between the chip 3502 and the cover 3501. Thechip or substrate 3502 may have a first face and a second face. Thefirst face may comprise a plurality of reservoirs 3508 configured tohold a liquid. The second face may comprise a plurality of channels. Thereservoirs 3508 may communicate with channels via through holes in thesubstrate 3502. A hydrophobic membrane 3503 may be sandwiched betweenthe chip 3502 and the cover 3501. The cover 3501 may be configured tocapture and compress two hydrophobic membrane filters 3503 that can actas valves that can allow air, but not fluid, to pass. When the cover3501 is assembled onto the microfluidic chip 3502, the compressiblegaskets 3504 on the underside of the cover 3501 (as shown in FIG. 35A)may provide a constant compressive force downward onto the chip 3502,thereby creating a seal to prevent or reduce leaking between channelsand/or the instrument. This downward force may initially be createdduring assembly with two sets of features between the chip 3502 and thecover 3501 engaging. First, the set of snaps 3505 around the cover mayengage with mating features 3506 on the chip 3502, thereby ensuringalignment between the cover 3501 and chip 3502 and providing initialcompression from the gasket feature onto the membrane 3503. Asadditional force is applied during assembly, the interference fitfeatures 3507 on the cover may engage with the external walls of alimited set of fluid reservoirs 3508 on the chip 3502. The interferencefit 3507 may be designed to maintain a fixed displacement, and thereforea fixed compressive force on the gasket 3504, when the assembly force isremoved. The fixed height standoffs 3509 adjacent to the interferencefeatures 3507 may limit the distance the cover 3502 can be pressed downonto the reservoirs 3508. The cover piece 3501 may comprise a matingsurface 3510 for interfacing with a pneumatic device, for example apneumatic manifold as described herein.

The chip 3502 may, for example, comprise any number of interference fitfeatures 3507 as desired by one of ordinary skill in the art. Forexample, the chip 3502 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 interference fit features 3507. Aswill be understood by one of ordinary skill in the art, the holdingcapacity of the interference fit features 3507 may depend on the numberof interference fit features 3507 on the chip 3502. For example, fourinterference fit features 3507 may have a holding capacity of 4 lb-f perfeature 3507 while eight interference fit features 35 o 7 may have aholding capacity of 2 lb-f per feature 3507 for the same decouplingforce. The interference fit features 3507 may have a radial interferencewithin a range of about 10 um to about 120 um, for example within arange of about 30 um to about 60 um, for example about 45 um.

FIGS. 36A-36B show an example of a fluidic device 3600 which comprisesmultiple parts. FIG. 36A shows an exploded perspective view of thethree-part fluidic device 3600. FIG. 36B shows an explodedcross-sectional view of the three-part fluidic device 3600. The fluidicdevice 3600 may comprise a cover piece or cover layer 3601, a chip plateor substrate 3602, a hydrophobic membrane 3603, and a compressiblegasket 3604. The hydrophobic membrane 3603 may comprise a strip ofhydrophobic membrane 3603 disposed within and/or across the pneumaticports 3605 and sandwiched between the cover 3601 and the substrate 3602.The compressible gasket 3604 may comprise a strip of gasket materialcomprising apertures which are shaped and spaced to correspond to thepneumatic ports 3605. The compressible gasket strip 3604 may besandwiched between the cover 3601, the hydrophobic membrane 3603, andthe chip 3602 to provide a constant compressive force on the chip 3602and create a seal to reduce or prevent leakage between channels. Thecover 3601 and the chip 3602 may comprise one or more mating features(e.g. snaps, interference fits, clamps, adhesives, screws, bolts, heightstandoffs, etc.) configured to couple the two pieces together asdescribed herein. The mating features may be configured to apply forceto the compressible gasket 3604 to seal the pneumatic ports 3605 asdescribed herein. The cover 3601 may be configured to interface with apneumatic device and/or other elements of an instrument, for example anyof the instruments described herein.

The pneumatic ports 3605 may comprise vertical cylindrical through-holesor apertures that extend from the pneumatic channels of the chip 3605and through the cover layer 3601. The portion of the pneumatic ports3605 on the chip layer 3602 may have a height at or near the height ofthe channels of the chip 3602. The pneumatic ports 3605 on the substrate3602 may be configured to have a height to minimize sample loss. Thepneumatic ports 3605 on the substrate 3602 may have a height within arange of about −1 mm to about 2 mm relative to a top surface of thesubstrate 3602, for example within a range of about −500 μm to about1000 μm relative to a top surface of the substrate 3602. One or more ofthe pneumatic ports 3605 may be inset from the top surface of thesubstrate 3602 (e.g. with a height within a range of about −1 um toabout −1 mm, or within a range of about −1 um to about −500 um relativeto the top surface). One or more of the pneumatic ports 3605 mayprotrude relatively perpendicularly from the top surface of thesubstrate 3602 (e.g. with a height within a range of about 0 um to about2 mm, or within a range of about 0 um to about 1000 um relative to thetop surface). The position of the hydrophobic membrane 3603 and gasket3604 within the pneumatic ports 3605 above the chip layer 3602 mayreduce or prevent liquid loss through the pneumatic ports 3605 whennegative pressure is applied thereto.

In some embodiments, the hydrophobic membrane 3603 and/or thecompressible gasket 3604 may be attached to the cover 3601 prior tointerlocking of the layers. Alternatively, the hydrophobic membrane 3603and/or the compressible gasket 3604 may be attached to the chip 3602prior to interlocking of the layers.

The chip 3602 may comprise any of the fluidic elements described herein.The chip 3602 may, for example, comprise a sample inlet reservoir 3606,a trailing electrolyte reservoir 3607, a leading electrolyte bufferingreservoir 3608, a leading electrolyte reservoir 3609, an elutionbuffering reservoir 3610, and an elution reservoir 3611 as describedherein. A first (e.g. top) face or side of the chip 3602 may comprisethe reservoirs 3606-3611 as shown. A second (e.g. bottom) face or sideof the chip 3602 may comprise one or more ITP channels (also referred toherein as fluidic circuits). The second side of the chip 3602 maycomprise any of the ITP channels, or any combination of ITP channels,described herein. For example, the second side of the chip 3602 maycomprise the 8 parallel channels shown in FIG. 38.

FIGS. 37A-37B show an example of a fluidic device 3700 which comprisesthree parts. FIG. 37A shows an exploded perspective view of thethree-part fluidic device 3700. FIG. 37B shows an explodedcross-sectional view of the three-part fluidic device 3700. The fluidicdevice 3700 may comprise a cover piece or cover layer 3701, a chip plateor substrate 3702, a hydrophobic membrane 3703, and a compressiblegasket 3704, which may be substantially similar to those of device 3600.The hydrophobic membrane 3703 may comprise a strip of hydrophobicmembrane 3703 disposed within and/or across the pneumatic ports 3705 andsandwiched between the cover 3701 and the substrate 3702. Thecompressible gasket 3704 may comprise a strip of gasket materialcomprising apertures which are shaped and spaced to correspond to thepneumatic ports 3705. Pneumatic ports 3705 may be substantially similarto those of device 3600. The cover 3701 and the chip 3702 may compriseone or more mating features (e.g. snaps, interference fits, heightstandoffs, etc.) configured to couple the two pieces together asdescribed herein. The mating features may be configured to apply forceto the compressible gasket 3704 to seal the pneumatic ports 3705 asdescribed herein. The cover 3701 may be configured to interface with apneumatic device and/or other elements of an instrument, for example anyof the instruments described herein. The device 3700 may furthercomprise a bottom layer of material 3706. The chip 3702 may bemanufactured such that three walls of the channels are formed on abottom layer or underside of the chip 3702. The bottom layer of material3706 may be coupled to the underside of the chip 3702 in order to formthe fourth wall of the channels, thereby creating closed channels. Thebottom layer of material 3706 may be coupled to the underside of thechip 3702 through the use of a solvent, heat, a solvent heat bond,pressure, adhesive bond, laser weld, or a combination thereof. Forexample, the material can be a heat seal which bonds to the chip surfacethrough application of heat which partially melts the materials, therebybonding them. In certain embodiments, bonding may be achieved throughthe use of a solvent which dissolves the materials, thereby causing themto flow together and bond.

In some embodiments, the bottom layer of material 3706 may comprise acyclic olefin copolymer as described herein. For example, the bottomlayer of material 3706 may comprise TOPAS® 8007.

In some embodiments, bonding of the bottom layer of material 3706 to theunderside of the chip 3702 may be achieved through the use of an organicsolvent, for example toluene.

FIG. 38 shows an exemplary channel schematic for 8 parallel channels onthe underside of the chip of a three part fluidic device 3800. Thedevice 3800 may comprise a multi-part device which may be substantiallysimilar to devices 3500, 3600, or 3700 described herein. The device 3800may comprise 8 parallel channels as described herein. Each channel maybe connected to a sample input well or reservoir 3801, an elutionreservoir 3802, an elution buffering reservoir 3803, a leadingelectrolyte reservoir 3804, a leading electrolyte buffering reservoir3805, and a trailing electrolyte reservoir 3806. Reservoirs 3801-3806may be coupled to the channel by through-holes or apertures as describedherein. Elution reservoir 3802 may be connected to elution bufferingreservoir 3803 by an elution buffering channel 3807. A capillary barrier3808 (e.g. a plateau capillary barrier as described herein) may beprovided in the elution buffering channel 3807 to reduce or preventmixing or pressure driven flow between the contents of the elutionbuffering reservoir 3803 and the elution reservoir 3802. Leadingelectrolyte reservoir 3804 may be connected to leading electrolytebuffering reservoir 3805 by a leading electrolyte buffering channel3809. A capillary barrier 3810 (e.g. a plateau capillary barrier) may beprovided in the leading electrolyte buffering channel 3809 to reduce orprevent mixing or pressure-driven flow between the contents of theleading electrolyte buffering reservoir 3805 and the leading electrolytereservoir 3804. Buffering reservoir 3803 may contain elution bufferelectrolytes at a higher ionic strength than those in elution reservoir3802, while buffering reservoir 3805 may contain leading electrolytes ata higher ionic strength than those in leading electrolyte reservoir3804. The device may further comprise pneumatic ports 3811 along itsedges which are configured to couple to a pneumatic device, for examplea vacuum source on a benchtop instrument. The pneumatic ports 3811 maybe coupled to the channels and reservoirs by gas channels 3812 asdescribed herein. Application of suction (i.e. negative pneumaticpressure) at the pneumatic ports 3811 may load the sample, leadingelectrolyte, and elution buffer into the channels. The gas channels 3812may be coupled to the channels at one or more capillary barriers suchthat the negative pressure is applied to said capillary barriers.Suction may be applied simultaneously or sequentially to the pneumaticports 3811 so as to load the channels simultaneously or in stages,respectively. The sample may be loaded into a first zone or sub-channelwhich extends from the trailing electrolyte reservoir 3806 to acapillary barrier 3813 at a 180° low dispersion turn in the channel. Thecapillary barrier 3813 may provide an interface between the sample andthe leading electrolyte buffer during loading so as to limit, reduce, orprevent mixing or pressure-driven flow. The capillary barrier 3813 maycomprise a cliff capillary barrier as described herein. The trailingelectrolyte reservoir 3806 may be connected to channel first zone orsub-channel by a trailing electrolyte channel 3814. A capillary barrier3815 (e.g. a cliff capillary barrier) may be provided in the trailingelectrolyte channel 3814 between the trailing electrolyte reservoir 3806and the first zone or sub-channel so as to limit, reduce, or preventmixing or pressure-driven flow between the contents of the trailingelectrolyte reservoir 3806 and the sample. The leading electrolyte maybe loaded into the second zone or sub-channel of the channel whichextends from capillary barrier 3813 to capillary barrier 3816. Thecapillary barrier 3816 (e.g. a plateau capillary barrier) may provide aninterface between the leading electrolyte buffer and the elution buffer.The first zone or sub-channel and the second zone or sub-channel maymake up an ITP branch of the fluidic channel or circuit. The elutionbuffer may be loaded into a third zone or sub-channel of channel whichextends from capillary barrier 3816 to elution reservoir 3802. The thirdzone or sub-channel may make up an elution branch of the fluidic channelor circuit.

Electrodes may for example be located in the trailing electrolytereservoir 3806, the leading electrolyte reservoir 3804, and/or theleading electrolyte buffering reservoir 3805 such that the electrodes donot directly contact sample material. The electrodes may be triggered toalter or control the applied electric field in response to feedback froma sensor, for example a voltage, current, conductivity, or temperaturesensor as described herein. For example, passage of the nucleic acidswithin the ITP zone from the second zone of channel to the third zone ofchannel may be detected and feedback from the detector may trigger theapplied current to change. The current may for example be increased,decreased, or ended according to the protocol of the instrument. Thecurrent may for example be paused (e.g. dropped temporarily to zero) inorder to enable on-chip quantification of the nucleic acids.Alternatively or in combination, the current may be decreased in orderto slow isotachophoresis within the third zone to allow the nucleicacids which may have dispersed upon transition from the leadingelectrolyte buffer to the elution buffer (or second leading electrolytebuffer) time to concentrate further before reaching the elution well3802.

Capillary barrier 3808 may comprise any capillary barrier desired by oneof ordinary skill in the art. For example, capillary barrier 3808 maycomprise a ramp capillary barrier, a plateau capillary barrier, or acliff capillary barrier.

Capillary barrier 3810 may comprise any capillary barrier desired by oneof ordinary skill in the art. For example, capillary barrier 3810 maycomprise a ramp capillary barrier, a plateau capillary barrier, or acliff capillary barrier.

Capillary barrier 3813 may comprise any capillary barrier desired by oneof ordinary skill in the art. For example, capillary barrier 3813 maycomprise a ramp capillary barrier, a plateau capillary barrier, or acliff capillary barrier.

Capillary barrier 3815 may comprise any capillary barrier desired by oneof ordinary skill in the art. For example, capillary barrier 3815 maycomprise a ramp capillary barrier, a plateau capillary barrier, or acliff capillary barrier.

Capillary barrier 3816 may comprise any capillary barrier desired by oneof ordinary skill in the art. For example, capillary barrier 3816 maycomprise a ramp capillary barrier, a plateau capillary barrier, or acliff capillary barrier.

FIG. 39 shows an exemplary multi-part device 3900 which may besubstantially similar to the multi-part devices described herein. Thedevice 3900 may comprise a cover layer 3901 and a chip or substrate 3902as described herein. The device 3900 may comprise a sample well 3903which may be connected to a sample channel via a through-hole oraperture as described herein. The sample well 3903 may comprise any ofthe sample wells described herein. Prior to use, the device 3900 maycomprise a sample seal layer 3904 configured to seal the sample well3903 prior to loading the sample, for example to enable pneumaticloading of one or more liquids from the other reservoirs into thechannel before the sample as described herein. The sample seal layer3904 may comprise a removable material. The sample seal layer 3904 maycomprise a heat-seal material or an adhesive material. The sample seallayer 3904 may comprise a thermoplastic film. The sample seal layer 3904may comprise a polymer or a plastic. The sample seal layer 3904 may, forexample, comprise a peelable polymer seal such as the 4titude® ClearHeat Seal Plus. The device 3900 may further comprise one or moreorienting features 3905 configured to prevent mis-insertion of thedevice 3900 into an instrument (e.g. any of the instruments describedherein). The orienting features 3905 may further prevent movement ofdevice 3900 after insertion into the instrument. The cover 3901 maycomprise a mating interface 3906 configured to interface with apneumatic device of the instrument, for example any of the instrumentsdescribed herein. The device 3900 may further comprise one or moreelements to facilitate use of the chip with the instrument, for examplea barcode tracking label 3907 configured to aid a user in tracking thedevice 3900 when in use as described herein.

Fluidic devices can be made from a variety of materials, including butnot limited to, glass (e.g., borosilicate glass), silicon, plastic, andelastomer. Plastics can include polymethylmethacrylate (PMMA), cyclicolefin copolymer (COC), cyclic olefin polymer (COP), polyethylene,polyethylene terephthalate (PET), high-density polyethylene (HDPE), andlow-density polyethylene (LDPE). Elastomers can includepolydimethylsiloxane (PDMS).

Any of the fluidic devices described herein may comprise a multi-partfluidic device. Multi-part fluidic devices may be made from one or morematerials described herein. In some embodiments, all of the parts of themulti-part fluidic devices described herein may be made from the samematerial(s). In some embodiments, one or more of the parts of themulti-part fluidic devices described herein may be made from the samematerial(s). In some embodiments, all of the parts of the multi-partfluidic devices described herein may be made from different material(s).

The cover piece may be made from a variety of materials, including butnot limited to, glass (e.g., borosilicate glass), silicon, plastic, andelastomer. Plastics can include polymethylmethacrylate (PMMA), cyclicolefin copolymer (COC), cyclic olefin polymer (COP), polyethylene,polyethylene terephthalate (PET), high-density polyethylene (HDPE), andlow-density polyethylene (LDPE). Elastomers can includepolydimethylsiloxane (PDMS).

The chip or substrate may be made from a variety of materials, includingbut not limited to, glass (e.g., borosilicate glass), silicon, plastic,and elastomer. Plastics can include polymethylmethacrylate (PMMA),cyclic olefin copolymer (COC), cyclic olefin polymer (COP),polyethylene, polyethylene terephthalate (PET), high-densitypolyethylene (HDPE), and low-density polyethylene (LDPE). Elastomers caninclude polydimethylsiloxane (PDMS). The chip or substrate may forexample comprise a COC such as TOPAS 8007.

The bottom layer may be made from a variety of materials, including butnot limited to, glass (e.g., borosilicate glass), silicon, plastic, andelastomer. Plastics can include polymethylmethacrylate (PMMA), cyclicolefin copolymer (COC), cyclic olefin polymer (COP), polyethylene,polyethylene terephthalate (PET), high-density polyethylene (HDPE), andlow-density polyethylene (LDPE). Elastomers can includepolydimethylsiloxane (PDMS). The chip or substrate may for examplecomprise a COC such as TOPAS 8007. In some embodiments, the bottom layercan comprise the same or a similar material as the chip. For example,both materials can have the same melting temperature.

The hydrophobic membrane may comprise an air-permeable hydrophobicmembrane. The hydrophobic membrane may not be liquid-permeable. Thehydrophobic membrane may be porous. The hydrophobic membrane maycomprise a flexible material such that it may be compressed by the coverand/or compressible gasket to seal the pneumatic ports and reduce orprevent fluid leakage as described herein.

The compressible gasket may be made from a variety of materials,including but not limited to, neoprene.

FIG. 40 shows an exemplary fluidic circuit 4000 comprising voltage andtemperature sensing. The fluidic circuit 4000 may be substantiallysimilar to the circuits described in FIG. 38. The fluidic circuit 4000may be comprise a channel connected to a sample input well or reservoir,an elution reservoir (EB), an elution buffering reservoir (EBH), aleading electrolyte reservoir (LE), a leading electrolyte bufferingreservoir (LEH), and a trailing electrolyte reservoir (TEH) as describedherein. The reservoirs 4001 may be positioned in the device (e.g. device3800) such that the wells 4001 are at standard locations for amicrotiter plate as described herein. Reservoirs 4001 may be coupled tothe channel by through-holes or apertures as described herein. Acapillary barrier (e.g. a plateau capillary barrier) may be provided inthe leading electrolyte buffering channel (between LE and LEH) to reduceor prevent mixing or pressure-driven flow between the contents of theleading electrolyte buffering reservoir (LEH) and the leadingelectrolyte reservoir (LE) as described herein. The device 4000 mayfurther comprise pneumatic ports 4002 along its edges which areconfigured to couple to a pneumatic device, for example a vacuum sourceon a benchtop instrument. The pneumatic ports 4002 may be positioned inthe device at standard locations for interfacing with commonly-availablepneumatic manifolds. The pneumatic ports 4002 may be coupled to thechannels and reservoirs by gas channels as described herein. Applicationof suction (i.e. negative pneumatic pressure) at the pneumatic ports4002 may load the sample, leading electrolyte, and elution buffer intothe channels as described herein. The gas channels 4002 may be coupledto the channels at one or more capillary barriers such that the negativepressure is applied to said capillary barriers as described herein.Suction may be applied simultaneously or sequentially to the pneumaticports 4002 so as to load the channels simultaneously or in stages,respectively. The sample may be loaded into a first zone or sub-channel4003 which extends from the trailing electrolyte reservoir (TEH) to acapillary barrier 4004 at a 180° low dispersion turn in the channel. Thecapillary barrier 4004 may provide an interface between the sample andthe leading electrolyte buffer during loading so as to limit, reduce, orprevent mixing or pressure-driven flow. The capillary barrier 4004 maycomprise a cliff capillary barrier as described herein. The capillarybarrier 4004 may enable bubble-free priming or loading of the sample andelution buffer within the channel 4000 as described herein. Thecapillary barrier 4004 may be used for feedback triggering as describedherein. For example, when the ITP band passes the capillary barrier4004, the derivative of the voltage may exhibit a peak as shown in FIG.92B. This peak may trigger the instrument to perform additional voltagesignal processing as described herein. The trailing electrolytereservoir (TEH) may be connected to channel first zone or sub-channel bya trailing electrolyte channel. A capillary barrier 4005 (e.g. a cliffcapillary barrier) may be provided in the trailing electrolyte channelbetween the trailing electrolyte reservoir (TEH) and the first zone orsub-channel 4003 so as to limit, reduce, or prevent mixing orpressure-driven flow between the contents of the trailing electrolytereservoir (TEH) and the sample as described herein. The leadingelectrolyte may be loaded into the second zone or sub-channel of thechannel which extends from capillary barrier 4004 to a capillary barrier4006 (e.g. a plateau capillary barrier) which may provide an interfacebetween the leading electrolyte buffer and the elution buffer. Anarrowing or construction 4007 may be provided within the second zone ofthe channel. The construction 4007 may be used for feedback triggeringas described herein. For example, when the ITP band passes theconstruction 4007, the derivative of the voltage may exhibit a peak asshown in FIG. 92D. This peak may trigger the instrument to performadditional signal processing (e.g. temperature signal processing) asdescribed herein. An integrated quantitation region 4008 may also beprovided within the second zone of the channel. The integratedquantitation region 4008 may be used to perform in-line (i.e. on-chip)quantitation of nucleic acids in the passing ITP band as describedherein. The first zone or sub-channel 4003 and the second zone orsub-channel may make up an ITP branch of the fluidic channel or circuit4000. The elution buffer may be loaded into a third zone or sub-channelof channel which extends from capillary barrier 4006 to the elutionreservoir (EB). The third zone or sub-channel may make up an elutionbranch of the fluidic channel or circuit 4000. The third zone maycomprise an infrared temperature sensor 4009 as described herein.

Capillary barrier 4004 may comprise any capillary barrier desired by oneof ordinary skill in the art. For example, capillary barrier 4004 maycomprise a ramp capillary barrier, a plateau capillary barrier, or acliff capillary barrier.

Capillary barrier 4005 may comprise any capillary barrier desired by oneof ordinary skill in the art. For example, capillary barrier 4005 maycomprise a ramp capillary barrier, a plateau capillary barrier, or acliff capillary barrier.

Capillary barrier 4006 may comprise any capillary barrier desired by oneof ordinary skill in the art. For example, capillary barrier 4006 maycomprise a ramp capillary barrier, a plateau capillary barrier, or acliff capillary barrier.

Materials used for the fabrication of fluidic devices can be selectedfor their optical properties. For example, materials can be used thatexhibit low auto-fluorescence, low scatter, and high transmission atwavelengths of interest (e.g., excitation and emission wavelengths fornucleic acid labels or dyes). Different materials can be used in onefluidic device; for example, a detection region can be fabricated withmaterials exhibiting useful optical properties, while other regions ofthe device can comprise other materials.

Materials used for the fabrication of fluidic devices can be selectedfor their thermal properties. For example, materials can be selected forhigh thermal conductivity. Alternatively, materials can be selected forlow thermal conductivity (e.g., to thermally insulate a fluidic deviceor a region of a fluidic device. Different materials can be used in onefluidic device; for example, a heating region can have materials withhigh thermal conductivity for improved thermal communication with athermal controller, while the heating region is surrounded by materialswith low thermal conductivity for thermal isolation from other regionsof the device.

Materials used for the fabrication of fluidic devices or microchannelstherein can be selected for their elastomeric or deformation properties.For example, materials can be selected for low elasticity so as to allowfor plastic channel closure as described herein. Alternatively,materials can be selected for high elasticity. Different materials canbe used in one fluidic device; for example poly(methyl methacrylate)(PMMA), cyclic olefin copolymer (COC), cyclo-olefin polymer (COP), orthe like can be used in a single fluidic device. Materials may have amodulus of elasticity of at least 1 GPa, 1.5 GPa, 2 GPa, 2.5 GPa, 3 GPa,3.5 GPa, 4 GPa, 4.5 GPa, or 5 GPa. Materials may have a modulus ofelasticity of at most 1 GPa, 1.5 GPa, 2 GPa, 2.5 GPa, 3 GPa, 3.5 GPa, 4GPa, 4.5 GPa, or 5 GPa. Materials may have a tensile strength of atleast 10 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90MPa, 100 MPa, 110 MPa, 120 MPa, 130 MPa, 140 MPa, 150 MPa, 160 MPa, 170MPa, 180 MPa, 190 MPa, 200 MPa. Materials may have a tensile strength ofat most 10 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa,90 MPa, 100 MPa, 110 MPa, 120 MPa, 130 MPa, 140 MPa, 150 MPa, 160 MPa,170 MPa, 180 MPa, 190 MPa, 200 MPa.

In some cases, surfaces of a fluidic device can be used without surfacetreatments or coatings. In other cases, surfaces of a fluidic device canbe used with surface coatings, such as hydrophobic treatments,hydrophilic treatments, or selective binding agents (e.g., antibodies).Different regions of a fluidic device can comprise different surfacetreatments (or the lack thereof). For example, some channels,reservoirs, or parts thereof can be hydrophobic, while others arehydrophilic.

Fluidic devices can include a range of flow control units andtechniques, including but not limited to capillary barriers, air outletreservoirs, gas/air lines, fill level monitors (e.g., by electrodemeasurement), particular reservoir geometries, particular fluidicresistances of channels, and fluid loading orders.

A capillary barrier is a constriction in the internal cross-sectionalarea of a fluidic channel that prevents flow of a liquid across thebarrier through capillary forces. The longitudinal shape of theconstriction can be abrupt or tapered. Capillary barrier shapes can takemany forms. However, three forms are particularly contemplated herein,“plateau” capillary barrier, “cliff” capillary barrier and “ramp”capillary barrier.

As used herein, a “plateau” capillary barrier comprises first and secondtapered areas, referred to as “ramps”, oriented in opposite directionsand typically separated by a plateau at which the internalcross-sectional area of the channel remains substantially the same.Accordingly, in a plateau barrier, the internal cross-sectional area ofthe channel experiences a decrease followed by an increase. The shapesof the ramps and the plateau can be flat (linear), such as a plane orwedge, or curved (non-linear), such as a hump, and can have alongitudinal shape of a circle, ellipse or parabola, to name a few. Alinear shape allows for equal pressure to move liquid across the barrierin a linear fashion. The angle between the base of the ramp to the pointof greatest constriction can be, for example, between about 25 degreesand about 70 degrees, e.g., between about 30 degrees to 45 degrees. Theinternal cross-sectional diameter of the channel at the base of the rampcan be between about 50 microns to 2000 microns (e.g., 400 microns to1200 microns). The internal cross-sectional diameter of the channel atthe top of the ramp or plateau can be between about 10 microns to about300 microns. The ratio between the two can be 2×. The plateau can serveto separate the meniscus of a liquid on the first side of the barrierfrom touching the meniscus of a liquid on the second side of the barrieruntil sufficient pressure is applied, such as negative pressure appliedbetween the two menisci.

The “cliff” capillary barrier comprises a first tapered area and acliff, typically separated by a plateau. The first tapered area andplateau can have shapes and dimensions as described for the plateaucapillary barrier. The cliff creates an abrupt change in the internalcross-sectional area of the channel. Typically, the cliff takes theshape of a steep wall, which can be flat or curved, and which rises atan angle from the floor of the channel at an angle of about 80 degreesto about 100 degrees, e.g., about 90 degrees. The plateau can be presentfor ease of manufacturing, by avoiding sharp angles.

The “ramp” capillary barrier, as used herein, comprises a first taperedarea and a cliff. Optionally, a “ramp” capillary barrier may include aplateau. The first tapered area and the plateau, if present, can haveshapes and dimensions as described for the plateau capillary barrier.The cliff creates an abrupt change in the internal cross-sectional areaof the channel. Typically, the cliff takes the shape of a steep wall,which can be flat or curved, and which rises at an angle from the floorof the channel at an angle of about 80 degrees to about 100 degrees,e.g., about 90 degrees. The plateau can be present for ease ofmanufacturing, by avoiding sharp angles.

The term capillary barrier refers to a constriction in a fluidic channelthat prevents or restricts fluid flow across the constriction bycapillary forces. Typically, the constriction is formed from an objectdisposed in the channel. A capillary barrier may be breached byapplication of a minimum positive or negative pressure to the fluidwhose flow is restricted by the barrier. Certain embodiments ofcapillary barriers are described in Patent Application Publication US2016/0160208 (Santiago). Capillary barriers can be paired with airoutlet reservoirs to purge air (e.g., to prevent bubbles), therebypositioning and successfully establishing a liquid-liquid interface (i)between leading and trailing electrolyte solutions that is required forisotachophoresis, and (ii) between buffering reservoirs and leadingelectrolyte or trailing electrolyte and/or sample solutions. Capillarybarriers can be designed in combination with channel geometry toautomate filling of channels in a preferred order. Channel resistancescan be selected, such as by design of channel dimensions, to providedifferential fluidic resistances. Ordering of liquid loading can allowthe correct formation of liquid-liquid interfaces without air bubblesfor performing electrokinetic processes. In one example, a trailing ionreservoir is directly connected to the analyte or sample channel.

Capillary barriers may be used to position a meniscus of a fluid at afluid-interface region using capillary forces. The capillary barrier mayact as a passive stopping mechanism and utilize surface forces to holdor pin the liquid meniscus of a fluid in a desired and stationarylocation. Once the meniscus of the fluid is pinned at a junction,different liquids may be loaded and backfilled to the meniscus of thefirst fluid to create a liquid-to-liquid interface. A capillary barriermay for example comprise a constriction or expansion (e.g., a change incross-sectional area) within the channel. Fluid flow within the channelmay rely in part on capillary forces, which may result from the surfacetension forces between the fluid and the channel walls. The magnitude ofthe capillary forces may be determined by the contact angle between thefluid and the channel walls. A capillary barrier may be configured tointroduce an abrupt change in the capillary force the liquid experienceswhile flowing through the channel, for example by changing an effectivediameter of the channel (either enlarging or narrowing the channel). Thechange in the capillary forces may be proportional to the difference inthe surface tensions of the fluid on each side of the capillary barrier.Fluid flow may be arrested by the capillary barrier due to surfaceforces caused by the change in cross-sectional area within the channel.Fluid at the cross-sectional area change may face an energetic barrierassociated with the different surface tensions on each side of thecapillary barrier.

As used herein, the term “burst pressure” refers to the minimum pressurerequired to move a meniscus of a stopped liquid over a capillarybarrier.

A “ramp capillary barrier”, for example as described in PatentApplication Publication US 2016/0160208 (Santiago), may comprise a rampand a cliff without a plateau region therebetween.

FIGS. 41A-41B show an exemplary “cliff capillary barrier” 4110. FIG. 41Ashows a top view of a channel 4100 having a cliff capillary barrier 4110disposed therein. FIG. 41B shows a longitudinal cross-sectional sideview of the cliff capillary barrier 4110 in the channel 4100. The cliffcapillary barrier 4110 may comprise a trapezoidal cross-section having aconstriction within the channel 4100 formed by an angled surface 4111and a plateau surface 4112 of the cliff capillary barrier 4110 followedby a sudden expansion within the channel formed by a cliff surface 4113.The channel 4100 may comprise a first wall 4101, a second wall 4102, athird wall 4103, and a fourth wall 4104 to form a closed channel. Thechannel 4100 may for example have a square or rectangular cross-section(taken along a lateral axis of the channel 4100) comprising four walls.The first and the third walls 4101, 4103 may be substantially parallelto one another. The second and the fourth walls 4102, 4104 may besubstantially parallel to one another. The cliff capillary barrier 4110may protrude from the second channel wall 4102 into the channel 4100.The cliff capillary barrier 4110 may be disposed on the second wall4102. Alternatively, the cliff capillary barrier 4110 may form a part ofthe second wall 4102. The cliff capillary barrier 4110 may comprisesides that are disposed on, coextensive with, or integrated in aninterior surface of the second wall 4102. The cliff capillary barrier4110 may extend substantially the width of the channel 4100. Forexample, the cliff capillary barrier 4110 may extend substantiallybetween the first and third walls 4101, 4103 as shown in FIG. 41A. Thecliff capillary barrier 4110 may comprise a first and a second lateralwall or side 4114, 4115. The first and second lateral walls or sides4114, 4115 may be connected to the first and third channel walls 4101,4103, respectively. Alternatively, the first and second lateral walls orsides 4114, 4115 may be coextensive with the first and third channelwalls 4101, 4103, respectively. Alternatively, the first and secondlateral walls or sides 4114, 4115 may be adjacent to the first and thirdchannel walls 4101, 4103, respectively. The first and second lateralwalls or sides 4114, 4115 may each comprise a cross-sectional area witha trapezoidal shape (for example the cross-sectional area shown in FIG.41B). The trapezoidal cross-section may comprise a plateau surface orside 4112 that is substantially parallel to the second channel wall4102. The plateau surface or side 4112 may be situated in the channel4100 between the second and fourth channel walls 4102, 4104. An angledsurface or side (also referred to herein as a ramp) 4111 may connect thesecond wall 4102 to the plateau surface or side 4112 at a first edge4116. A cliff surface or side 4113 may connect the second wall 4102 tothe plateau surface or side 4112 at a second, opposite edge 4117.

The angled surface or side 4111 may be configured to gradually reducethe height of the channel 4100 from a first height h₁ to a second,smaller height h₂, over a distance along the length of the channel. Thefirst height h₁ may be at least twice as large as the second height h₂.The angled surface or side 4111 may for example be an incline planerising from a bottom wall of the channel 4100 or a decline planelowering from a top wall of the channel 4100. The angled surface or side4111 may for example be an angled plane extending into the channel 4100from a side wall of the channel 4100. The angled surface or side 4111may have a first edge 4116 which intersects with the plateau region orside 4112 to form an interior obtuse angle of the cliff capillarybarrier and a second, opposing edge 4118 which intersects with thesecond channel wall 4102 to form an interior acute angle θ of the cliffcapillary barrier 4110.

The cliff surface or side 4113 may be configured to suddenly increasethe height of the channel 4100 from a first height h₂ to a second,larger height h₃, over a very short distance or no distance along thelength of the channel 4100. The cliff surface or side 4113 may forexample be a vertical surface (relative to the second wall 4102)connecting the plateau surface or side 4112 to the second wall 4102. Thecliff surface or side 4113 may for example be substantiallyperpendicular to the second wall 4102.

Liquid wicking up the angled surface or side to the plateau surface orside 4111 may face an energetic barrier associated with expanding pastthe plateau surface or side 4112 (as additional liquid surface area orpressure is required to advance the liquid) which may result in theliquid being stopped by the cliff capillary barrier 4110 and a meniscusof the liquid being positioned at the edge 4116 of the plateau surfaceor side 4112 nearest the angled surface or side 4111 or the edge 4116above the cliff surface or side 4113. The cliff capillary barrier 4110may be configured such that the liquid stopped by the capillary barrier4110 can be wetted by liquid approaching the cliff capillary barrier4110 from its other side (e.g. from the cliff side 4113) to create abubble-free liquid-to-liquid interface. The cliff capillary barrier 4110may be disposed adjacent a gas channel 4120 configured to facilitate airbubble removal from the channel 4100 as the liquid enters the channel4100 and the meniscus of the liquid is stopped at the cliff capillarybarrier 4110 as described herein.

The cliff capillary barrier 4110 may be configured to hold the menisciof the liquids on either side of the cliff capillary barrier 4110separate, with an air gap between them spanning the plateau surface orside 4112 until a pressure applied across the capillary barrier via theair channel 4120 exceeds the burst pressure of the cliff capillarybarrier 4110 and one or both of the liquids cross the plateau surface orside 4112 to meet each other and form a liquid-to-liquid interface asdescribed herein (e.g., as shown in FIG. 47A).

The cliff capillary barrier 4110 may be configured to hold or stop aliquid when a pneumatic pressure is applied thereto. The cliff capillarybarrier 4110 may be configured to hold the liquid under a pressurewithin a range of about 0 mpsi to about 200 mpsi, for example within arange of about 10 mpsi to about 80 mpsi. The cliff capillary barrier4110 may be configured to hold the liquid until a burst pressure (e.g.the minimum pressure required to move the stopped liquid over plateau4112 and/or the cliff 4113 and past the cliff capillary barrier 4110) isreached. It will be understood by one of ordinary skill in the art thatthe burst pressure of the cliff capillary barrier 4110 may depend on theliquid(s) being held by the cliff capillary barrier 4110, with morewetting liquids having a lower burst pressure than less wetting liquids.

The angled surface or side 4111 may be configured to gradually reducethe height of the channel 4100 from a first height h₁ within a range ofabout 50 um to about 2 mm to a second height h₂ within a range of about10 um to about 30 um. The first height h₁ may for example be within arange of about 400 um to about 1.2 um.

The angled surface or side 4111 may have a first edge 4116 whichintersects with the plateau region or side 4112 to form an interiorobtuse angle of the cliff capillary barrier 4110.

The angled surface or side 4111 may have a second, opposing edge 4118which intersects with the second channel wall 4102 to form an interioracute angle θ of the cliff capillary barrier 4110. The interior acuteangle θ may be within a range of about 0 degrees to about 70 degrees,for example within a range of about 30 degrees to about 45 degrees orwithin a range of about 30 degrees to about 60 degrees.

The plateau surface or side 4112 may have a length along a longitudinalaxis of the channel 4110 within a range of about 500 um to about 1 mm,for example about 750 um.

The cliff surface or side 4113 may be substantially perpendicular to thesecond channel wall 4102 and/or the plateau surface or side 4112. Thecliff surface or side 4113 may intersect the second channel wall 4102 toform an interior angle φ within a range of about 60 degrees to about 90degrees.

The ramp 4111, plateau area 4112, or cliff area 4113, in anycombination, may have a substantially flat surface.

The ramp 4111, plateau area 4112, or cliff area 4113, in anycombination, may have a curved surface.

The ramp 4111, plateau area 4112, or cliff area 4113, in anycombination, may have a surface that comprises one or more grooves,ridges, indentations, steps, etchings, or protrusions.

The ramp 4111, plateau area 4112, or cliff area 4113, in anycombination, may have a surface that comprises regions with faces atdifferent angles.

The cliff capillary barrier 4110 may be made from a variety ofmaterials, including but not limited to, glass (e.g., borosilicateglass), silicon, plastic, and elastomer. Plastics can includepolymethylmethacrylate (PMMA), cyclic olefin copolymer (COC), cyclicolefin polymer (COP), polyethylene, polyethylene terephthalate (PET),high-density polyethylene (HDPE), and low-density polyethylene (LDPE).Elastomers can include polydimethylsiloxane (PDMS). The chip orsubstrate may for example comprise a COC such as TOPAS 8007. The cliffcapillary barrier 4110 may be made from the same material(s) as thechannel or a different material(s) as the channel 4100.

The depth of the channels 4100 on either side of the cliff capillarybarrier 4110 may be the same. Alternatively, each side 4111, 4113 of thecliff capillary barrier 4110 may be coupled to channels 4110 ofdifferent depths. For example, the ramp portion 4111 of the cliffcapillary barrier 4110 may be coupled to a sample channel 4105comprising a depth within a range of about 10 um to about 2 mm, forexample within a range of about 400 um to about 1.2 mm as describedherein. The cliff portion 4113 of the cliff capillary barrier 4110 maybe coupled to a leading electrolyte channel 4106 comprising a depthwithin a range of about 10 um to about 1 mm, for example within a rangeof about 10 um to about 600 um as described herein.

So, for example, referring to FIG. 41B, when the elements are positionedadjacently as ramp-plateau-cliff 4111-4112-4113, the cliff capillarybarrier 4110 can comprise a ramp 4111 rising from a surface 4102 of thechannel 4100 at a shallow angle θ, a plateau area 4112 having a surfaceabout parallel to other portions of the channel surface 4102, 4104, anda cliff 4113 falling to the surface 4102 and having an angle φsubstantially steeper than the angle θ of the ramp 4111. The shallowangle θ can be less than 60 degrees, e.g., no more than 45 degrees or nomore than 30 degrees. The cliff angle φ can be greater than 60 degrees,e.g., about 90 degrees. The plateau 4112 can be no more than 10 degreesoff parallel to the channel surface 4102.

FIGS. 42A-42B show an exemplary “plateau capillary barrier” 4210. FIG.42A shows a top view of a channel 4200 having a plateau capillarybarrier 4210 disposed therein. FIG. 42B shows a longitudinalcross-sectional side view of the plateau capillary barrier 4210 in thechannel 4200. The plateau capillary barrier 4210 may comprise atrapezoidal cross-section having a constriction within the channel 4200formed by a first angled surface 4211 and a plateau surface 4212 of theplateau capillary barrier 4210 followed by a gradual expansion withinthe channel 4200 formed by a second angled surface 4213. The channel4200 may comprise a first wall 4201, a second wall 4202, a third wall4203, and a fourth wall 4204 to form a closed channel. The channel 4200may for example have a square or rectangular cross-section (taken alonga lateral axis of the channel 4200) comprising four walls. The first andthe third walls 4201, 4203 may be substantially parallel to one another.The second and the fourth walls 4202, 4204 may be substantially parallelto one another. The plateau capillary barrier 4210 may protrude from thesecond channel wall 4202 into the channel 4200. The plateau capillarybarrier 4210 may be disposed on the second wall 4202. Alternatively, theplateau capillary barrier 4210 may form a part of the second wall 4202.The plateau capillary barrier 4210 may comprise sides that are disposedon, coextensive with, or integrated in an interior surface of the secondwall 4202. The plateau capillary barrier 4210 may extend substantiallythe width of the channel 4200. For example, the plateau capillarybarrier 4210 may extend substantially between the first and third walls4101, 4013 as shown in FIG. 42A. The plateau capillary barrier 4210 maycomprise a first and a second lateral wall or side 4214, 4215. The firstand second lateral walls or sides 4214, 4215 may be connected to thefirst and third channel walls 4201, 4203, respectively. Alternatively,the first and second lateral walls or sides 4214, 4215 may becoextensive with the first and third channel walls 4201, 4203,respectively. Alternatively, the first and second lateral walls or sides4214, 4215 may be adjacent to the first and third channel walls 4201,4203, respectively. The first and second lateral walls or sides 4214,4215 may each comprise a cross-sectional area with a trapezoidal shape(for example the cross-sectional area shown in FIG. 42B). Thetrapezoidal cross-section may comprise a plateau surface or side 4212that is substantially parallel to the second channel wall 4202. Theplateau surface or side 4212 may be situated in the channel 4200 betweenthe second and fourth channel walls 4202, 4204. A first angled surfaceor side 4211 (also referred to herein as a ramp) may connect the secondwall 4202 to the plateau surface or side 4212 at a first edge. A secondangled surface or side 4213 may connect the second wall 4204 to theplateau surface or side 4212 at a second, opposite edge 4217.

The first angled surface or side 4211 may be configured to graduallyreduce the height of the channel 4200 from a first height h₄ to asecond, smaller height h₅, over a distance along the length of thechannel 4200. The first height h₄ may be at least twice as large as thesecond height h₅. The first angled surface or side 4211 may for examplebe an incline plane rising from a bottom wall of the channel 4200 or adecline plane lowering from a top wall of the channel 4200. The firstangled surface or side 4211 may for example be an angled plane extendinginto the channel 4200 from a side wall of the channel 4200. The firstangled surface or side 4211 may have a first edge 4216 which intersectswith the plateau region or side 4212 to form an interior obtuse angle ofthe plateau capillary barrier 4210 and a second, opposing edge 4218which intersects with the second channel wall 4202 to form an interioracute angle α of the plateau capillary barrier 4210.

The second angled surface or side 4213 may be configured to graduallyincrease the height of the channel 4200 from a first height h₅ to asecond, larger height h₆, over a distance along the length of thechannel 4200. The first height h₅ may be at least twice as small as thesecond height h₆. The second angled surface or side 4213 may for examplebe a decline plane lowering from a bottom wall of the channel 4200 or anincline plane rising from a top wall of the channel 4200. The secondangled surface or side 4213 may for example be an angled plane extendingtowards a side wall of the channel 4200 from the plateau surface or side4212. The second angled surface or side 4213 may have a first edge 4217which intersects with the plateau region or side 4212 to form aninterior obtuse angle of the plateau capillary barrier 4210 and asecond, opposing edge 4219 which intersects with the second channel wall4202 to form an interior acute angle β of the plateau capillary barrier4210.

Liquid wicking up the first angled surface or side 4211 to the plateausurface or side 4212 may face an energetic barrier associated withexpanding past the plateau surface or side 4212 (as additional liquidsurface area or pressure is required to advance the liquid) which mayresult in the liquid being stopped by the plateau capillary barrier 4210and a meniscus of the liquid being positioned at the edge 4216 of theplateau surface or side 4212 nearest the first angled surface or side4211 or the edge 4217 above the second angled surface or side 4213. Theplateau capillary barrier 4210 may be configured such that the liquidstopped by the plateau capillary barrier 4210 can be wetted by liquidapproaching the plateau capillary barrier 4210 from its other side (e.g.from the second angled side) to create a bubble-free liquid-to-liquidinterface. The plateau capillary barrier 4210 may be disposed adjacent agas channel 4220 configured to facilitate air bubble removal from thechannel 4200 as the liquid enters the channel 4200 and the meniscus ofthe liquid is stopped at the plateau capillary barrier 4210 as describedherein.

The plateau capillary barrier 4210 may be configured to hold the menisciof the liquids on either side of the plateau capillary barrier 4210separate, with an air gap between them spanning the plateau surface orside 4212 until a pressure applied across the capillary barrier 4210 viathe air channel 4220 exceeds the burst pressure of the plateau capillarybarrier 4210 and one or both of the liquids cross the plateau surface orside 4212 to meet each other and form a liquid-to-liquid interface asdescribed herein (e.g., as shown in FIG. 47A).

The plateau capillary barrier 4210 may be configured to hold or stop aliquid when a pneumatic pressure is applied thereto. The plateaucapillary barrier 4210 may be configured to hold the liquid under apressure within a range of about 0 mpsi to about 200 mpsi, for examplewithin a range of about 10 mpsi to about 80 mpsi. The plateau capillarybarrier 4210 may be configured to hold the liquid until a burst pressure(e.g. the minimum pressure required to move the stopped liquid overplateau 4112 and/or onto the second angled region 4213 and past theplateau capillary barrier 4210) is reached. It will be understood by oneof ordinary skill in the art that the burst pressure of the plateaucapillary barrier 4210 may depend on the liquid(s) being held by theplateau capillary barrier 4210, with more wetting liquids having a lowerburst pressure than less wetting liquids.

In some embodiments, the burst pressure of the cliff capillary barrier4110 may be the same as the burst pressure of the plateau capillarybarrier 4210.

In some embodiments, the burst pressure of the cliff capillary barrier4110 may be higher than the burst pressure of the plateau capillarybarrier 4210. The higher burst pressure of the cliff capillary barrier4110 may facilitate loading (and stopping) of liquids which have lowersurface tensions, for example liquids comprising one or more surfactantsor detergents. For example, the sample may have a low enough surfacetension so as to wet across a plateau capillary barrier 4220 under thenegative pneumatic pressure applied by the instrument to the channel. Insuch case, the sample may be bounded within the channel by cliffcapillary barriers 4110 (e.g. a first cliff capillary barrier 4110between the sample and the LE and a second cliff capillary barrier 4110between the sample and the TE as described herein) so as to hold thesample in the channel during loading of the chip.

The first angled surface or side 4211 may be configured to graduallyreduce the height of the channel 4200 from a first height h₄ within arange of about 50 um to about 2 mm to a second height h₅ within a rangeof about 10 um to about 30 um. The first height h₄ may for example bewithin a range of about 400 um to about 1.2 um.

The first angled surface or side 4211 may have a first edge 4216 whichintersects with the plateau region or side 4212 to form an interiorobtuse angle of the cliff capillary barrier 4210.

The first angled surface or side 4211 may have a second, opposing edge4218 which intersects with the second channel wall 4202 to form aninterior acute angle α of the plateau capillary barrier 4210. Theinterior acute angle α may be within a range of about 0 degrees to about70 degrees, for example within a range of about 30 degrees to about 45degrees or within a range of about 30 degrees to about 60 degrees.

The plateau surface or side 4212 may have a length along a longitudinalaxis of the channel within a range of about 500 um to about 1 mm, forexample about 750 um.

The second angled surface or side 4213 may be configured to graduallyincrease the height of the channel from a first height h₅ within a rangeof about 10 um to about 30 um to a second height h₆ within a range ofabout 50 um to about 2 mm. The first height h₅ may for example be withina range of about 400 um to about 1.2 um.

The second angled surface or side 4213 may have a first edge 4217 whichintersects with the plateau region or side 4212 to form an interiorobtuse angle of the plateau capillary barrier 4210.

The second angled surface or side 4213 may have a second, opposing edge4219 which intersects with the second channel wall 4202 to form aninterior acute angle β of the plateau capillary barrier 4210. Theinterior acute angle β may be within a range of about 0 degrees to about70 degrees, for example within a range of about 30 degrees to about 45degrees or within a range of about 30 degrees to about 60 degrees.

The first angled surface 4211 (i.e. ramp), plateau area 4212, or secondangled surface area 4213, in any combination, may have a substantiallyflat surface.

The first angled surface 4211 (i.e. ramp), plateau area 4212, or secondangled surface area 4213, in any combination, may have a curved surface.

The first angled surface 4211 (i.e. ramp), plateau area 4212, or secondangled surface area 4213, in any combination, may have a surface thatcomprises one or more grooves, ridges, indentations, steps, etchings, orprotrusions.

The first angled surface 4211 (i.e. ramp), plateau area 4212, or secondangled surface area 4213, in any combination, may have a surface thatcomprises regions with faces at different angles.

So, for example, referring to FIG. 42B, the ramp barrier can comprisetwo ramps separated by a plateau. A first ramp 4211 can rise from asurface of the channel 4202 at a shallow angle α, a plateau area 4212can be about parallel to the channel 4200 and a second ramp 4213 canfall to the channel surface 4202 at a shallow angle β. The shallowangles α, β can be no more than 60 degrees, no more than 45 degrees orno more than 30 degrees. The shallow angles α, β can be the same angleor different angles.

The plateau capillary barrier 4210 may be made from a variety ofmaterials, including but not limited to, glass (e.g., borosilicateglass), silicon, plastic, and elastomer. Plastics can includepolymethylmethacrylate (PMMA), cyclic olefin copolymer (COC), cyclicolefin polymer (COP), polyethylene, polyethylene terephthalate (PET),high-density polyethylene (HDPE), and low-density polyethylene (LDPE).Elastomers can include polydimethylsiloxane (PDMS). The chip orsubstrate may for example comprise a COC such as TOPAS 8007. The plateaucapillary barrier 4210 may be made from the same material(s) as thechannel 4200 or a different material(s) as the channel 4200.

The depth of the channels 4200 on either side of the plateau capillarybarrier 4210 may be the same. Alternatively, each side of the plateaucapillary barrier 4210 may be coupled to channels 4200 of differentdepths as described herein.

FIG. 43 shows an exemplary channel or fluidic circuit 4300 highlightingthe initial fluid interface positions after loading. The fluidic circuit4300 may be substantially similar to the circuits described in FIG. 38.The fluidic circuit 4300 may be connected to a sample input well orreservoir 4301, an elution reservoir 4302, an elution bufferingreservoir 4303, a leading electrolyte reservoir 4304, a leadingelectrolyte buffering reservoir 4305, and a trailing electrolytereservoir 4306. Reservoirs 4301-4306 may be coupled to the channel bythrough-holes or apertures as described herein. Elution reservoir 4302may be connected to elution buffering reservoir 4303 by an elutionbuffering channel 4307. A capillary barrier 4308 (e.g. a plateaucapillary barrier as described herein) may be provided in the elutionbuffering channel 4307 to reduce or prevent mixing or pressure drivenflow between the contents of the elution buffering reservoir 4303 andthe elution reservoir 4302. Leading electrolyte reservoir 4304 may beconnected to leading electrolyte buffering reservoir 4305 by a leadingelectrolyte buffering channel 4309. A capillary barrier 4310 (e.g. aplateau capillary barrier) may be provided in the leading electrolytebuffering channel 4309 to reduce or prevent mixing or pressure-drivenflow between the contents of the leading electrolyte buffering reservoir4305 and the leading electrolyte reservoir 4304. Buffering reservoir4303 may contain elution buffer electrolytes at a higher ionic strengththan those in elution reservoir 4302, while buffering reservoir 4305 maycontain leading electrolytes at a higher ionic strength than those inleading electrolyte reservoir 4304. The device may further comprisepneumatic ports 4311 along its edges which are configured to couple to apneumatic device, for example a vacuum source on a benchtop instrument.The pneumatic ports 4311 may be coupled to the channels and reservoirsby gas channels 4312 as described herein. Application of suction (i.e.negative pneumatic pressure) at the pneumatic ports 4311 may load thesample, leading electrolyte, and elution buffer into the channels. Thegas channels 4312 may be coupled to the channels at one or morecapillary barriers such that the negative pressure is applied to saidcapillary barriers. Suction may be applied simultaneously orsequentially to the pneumatic ports 4311 so as to load the channelssimultaneously or in stages, respectively. The sample may be loaded intoa first zone or sub-channel which extends from the trailing electrolytereservoir 4306 to a capillary barrier 4313 at a 180° low dispersion turnin the channel. The capillary barrier 4313 may provide an interfacebetween the sample and the leading electrolyte buffer during loading soas to limit, reduce, or prevent mixing or pressure-driven flow. Thecapillary barrier 4313 may comprise a cliff capillary barrier asdescribed herein. The trailing electrolyte reservoir 4306 may beconnected to channel first zone or sub-channel 4317 by a trailingelectrolyte channel 4314. A capillary barrier 4315 (e.g. a cliffcapillary barrier) may be provided in the trailing electrolyte channel4314 between the trailing electrolyte reservoir 4306 and the first zoneor sub-channel 4317 so as to limit, reduce, or prevent mixing orpressure-driven flow between the contents of the trailing electrolytereservoir 4306 and the sample. The leading electrolyte may be loadedinto the second zone or sub-channel 4318 of the channel 4300 whichextends from capillary barrier 4313 to capillary barrier 4316. Thecapillary barrier 4316 (e.g. a plateau capillary barrier) may provide aninterface between the leading electrolyte buffer and the elution buffer.The first zone or sub-channel 4317 and the second zone or sub-channel4318 may make up an ITP branch of the fluidic channel or circuit. Theelution buffer may be loaded into a third zone or sub-channel 4319 ofchannel 4300 which extends from capillary barrier 4316 to elutionreservoir 4302. The third zone or sub-channel 4319 may make up anelution branch of the fluidic channel or circuit. Upon loading thefluids into the channel, the interfaces between liquids may be locatedabove or situated at their respective capillary barriers. The interfacebetween the trailing electrolyte buffer and the sample may be situatedat cliff capillary barrier 4315. The interface between the leadingelectrolyte buffer and the sample may be situated at cliff capillarybarrier 4313. The interface between the leading electrolyte buffer andthe elution buffer may be situated at ramp capillary barrier 4316. Theinterface between the leading electrolyte buffer and the highconcentration leading electrolyte buffer may be situated at rampcapillary barrier 4310. The interface between the high concentrationelution buffer and the elution buffer may be situated at ramp capillarybarrier 4308.

FIG. 44 shows an exemplary channel or fluidic circuit 4300 highlightingthe final fluid interface positions after loading. After loading thevarious buffers into the channel 4300, one or more fluid interfaceformed between the buffers may be flowed within the channel to move theinterface away from the capillary barrier(s) which formed the fluidinterface(s).

Moving an interface may reduce or minimize retention of nucleic acids ata capillary barrier, particularly between the leading electrolyte bufferand the elution buffer. Not wanting to be limited by a particulartheory, it is believed that this may help to maintain the DNA in a morecompact state as it passes through a constricted space of the capillarybarrier (e.g. a cliff capillary barrier 4316 at the junction between theLE and elution buffer) which may otherwise impair passage of a moredispersed ITP band. For example, the interface 4402 between the leadingelectrolyte buffer and the elution buffer may be moved downstreamtowards the elution reservoir 4302 such that the cliff capillary barrier4316 is fully engulfed by the leading electrolyte buffer when theinterface 4402 is arrested within a zone 4401. When flow of theinterface 4402 is arrested, the DNA may pass through the constrictedspace of the capillary barrier 4316 more easily in the leadingelectrolyte buffer than it would have had it passed through theinterface 4402 and the capillary barrier 4316 at the same time, as thetransition from the leading electrolyte buffer to the elution buffer mayhave cause the ITP band to expand, making it harder to pass over thebarrier.

Alternatively or in combination, moving an interface may reduce orminimize mixing of buffers and undesired contamination of buffers,particularly between the elution buffer and the high concentrationelution buffer or between the leading electrolyte buffer and the highconcentration leading electrolyte buffer. For example, the interface4403 between the elution buffer and the high concentration elutionbuffer may be moved downstream towards the elution buffering reservoir4303 in order to increase the distance between the interface 4403 andthe elution reservoir 4302 and reduce or minimize contamination of theelution buffer with the high concentration elution buffer, which couldnegatively affect the compatibility of the elution buffer withdownstream assays. Similarly, interface 4404 between the leadingelectrolyte buffer and the high concentration leading electrolyte buffermay be moved downstream towards the leading electrolyte bufferingreservoir 4305 in order to increase the distance between the interface4404 and the leading electrolyte reservoir 4304 and reduce or minimizecontamination of the leading electrolyte buffer with the highconcentration leading electrolyte buffer.

In some embodiments, one or more interfaces may be flowed away from itscorresponding capillary barrier by applying a negative pressure to oneor more pneumatic ports of the channel.

Alternatively or in combination, the one or more interfaces may beflowed away from its corresponding capillary barrier due to gravity. Forexample, the liquid head heights in the reservoirs may be adjusted togenerate gravity-driven flow within the channel. After loading, thefluid pressures within the channel may be allowed to equilibrate suchthat one or more interfaces between the fluids in the channel flowswithin the channel towards its final fluid interface position. The finalposition of the fluid interface may be adjusted by adjusting therelative head heights of the fluids within the reservoirs as will beunderstood by one of ordinary skill in the art.

Gas (e.g., air) channels or lines can be used to provide actuatedpneumatic pressure to capillary barriers or other regions of a fluidicdevice. Gas channels can connect to external gas pressure sources viapneumatic ports. Gas channels can have higher fluidic resistance thanthe liquid channels they provide pressure to, for example to reduce orprevent liquid flow into the gas channel. For example, gas channels canhave less than half the cross sectional area of a main isotachophoresischannel. Multiple gas channels can be connected to a single gasreservoir or port (e.g., with branching channels). Capillary valves canbe employed with branched air lines to prevent upstream liquid movement.FIG. 10A shows an exemplary gas channel 1001 which may comprise acapillary barrier 1002 connected to the liquid channel interface 1003between the sample 1004 and leading electrolyte buffer 1005sub-channels. FIG. 10B is a magnified schematic of the gas channel 1001highlighting the capillary barrier 1002 which prevents upstream liquidmovement towards the pneumatic port 1006.

FIG. 45A shows an example of the fluidic layer of the chip. Pneumaticactuation channels (also referred to herein as air channels or airlines) 4501 in this example are narrow, shallow, and straight tominimize total volume. The minimum dimensions may be set bymanufacturing requirements or by the maximum allowable pressure dropacross these pneumatic channels. These dimensions are between 50 um and500 um for both the width and height of the channel. These pneumaticchannels lead to the pneumatic ports 4502. The ports are verticalcylindrical holes terminating at membrane rest plane. In comparison tothe pneumatic channels, the liquid channels on the chip layer are widerand deeper. This may allow a low fraction of the liquid to be drawn intothe pneumatic lines. The pneumatic channels 4501 may comprise aconstriction at the junctions between the pneumatic channels 4501 andthe liquid channel, which may act as a capillary barrier to reduce orprevent liquid from entering the pneumatic channels 4501. FIG. 45B showsa cutaway of the pneumatic port 4502. 4504 is the connection of the portto the pneumatic channel on the fluidic layer. 4503 is the membrane restplane. A hydrophobic membrane is applied to this surface and held inplace by a compressed gasket, by an adhesive, or by a thermal bond. Aircan pass this membrane, but liquid cannot. The diameter of this port iscontrolled by manufacturing requirements, and can have a diameter below1 mm. The height of the vertical cylinder should be below 2 mm. FIG. 45Cshows an implementation of this design. The hydrophobic membrane ismarked as 4505. The fluorescent liquid 4506 is confined to the verticalcolumn of the pneumatic port, but doesn't spread beyond. Pneumaticactuation can be used to move liquids in fluidic channels. When doingso, it can be valuable to minimize liquid loss into the pneumaticactuation channel, as this may be liquid volume that is not processed.This design is for a fluidic air port and channel design that minimizesloss by manipulating channel and port geometry, and including ahydrophobic membrane that prevents liquid transfer but allows airtransfer.

FIG. 46A shows an exemplary microfluidic channel in which an emptysample channel can be detected. The sample channel 4601 is terminated onboth sides 4602 by channels that may have liquid loaded prior to sampleloading (e.g. trailing electrolyte and leading electrolyte,respectively). This loading is subject to failure by the liquid movinginto the sample channel 4601, which is intended to be empty prior toloading. The sample channel 4601 is also connected on both ends topneumatic actuation ports 4603 that can be connected to a pressurecontrol circuit or allowed to vent to the atmosphere or a pneumaticmanifold as described herein. The arrows 4604 on the drawing show thedetection path, which passes from one pneumatic port 4603, through thesample channel 4601, and through the other pneumatic port 4603. FIG. 46Bshows the sequence of events for detection of whether this channel 4601is empty or filled. First the pneumatic control circuit is set (Step4611) to achieve a target pressure, between 1 psig and −1 psig. Second,both of the valves connected to the channel are connected to thepneumatic control circuit (Step 4612). The pressure is allowed to returnto the control point if there is a disturbance caused by the valvesopening, then the signal of the pneumatic control circuit is read (Step4613). This signal may commonly be associated with the voltage appliedto a pneumatic proportional valve. After this, either one of the valvesconnected to the channel can be changed to a state where it vents thechannel to atmosphere. This allows air to flow through the detectionpath previously shown (Step 4614, e.g., FIG. 46A). This increased airflow will cause a change in the signal to the control circuit, commonlya change in voltage to further open the proportional valve allowing airflow. The pressure is once again allowed to settle, and the signal onthe control circuit is measured (Step 4615). The two measured controlsignals are subtracted and compared to a threshold (Step 4616) and thechannel is determined to be either empty or full (Step 4617). Afterthis, both sides of the channel may be returned to some default state,commonly by venting them to atmosphere (Step 4618). FIG. 46C showspressure traces and control signal traces from the implementation ofthis technique. On the top trace, the pressure reading, the settling topressure 4621 and the venting of the valves 4628 is visible. The controlsignal, below, does correspond to the voltage applied to a pneumaticproportional valve to regulate pressure. This shows settling to pressure4621, both valves opening 4622, the settled value after opening 4623, asingle valve closing 4624, the measurement of the signal 4625, and theventing of the channel 4628. This pneumatic control and detection schemeallows detection of a completely empty channel prior to a user loadingsample. This is useful in cases in which sample loading requires acompletely empty channel prior to loading for successful completion ofthe load. If a previous step has wetted the channel, this allows theuser to avoid sample loading in this channel, conserving sample. Thisdetection technique uses the same pneumatic detection and actuationhardware as pneumatic priming.

Negative pressure or vacuum can be applied to the gas channels via thegas ports in order to load a fluidic channel. Each fluidic channel on amicrofluidic device may be loaded simultaneously or independently (e.g.sequentially) of one another. Within a channel, the fluids may be loadedsimultaneously or independently of one another. For example, leadingelectrolyte buffer, high concentration leading electrolyte buffer,trailing electrolyte buffer, high concentration trailing electrolytebuffer, the elution buffer, high concentration elution buffer, or anycombination thereof may be loaded prior to, simultaneously with, orafter loading the sample. For example, negative pressure may be appliedto the gas ports on one side of the chip to load one or more fluids(e.g. trailing electrolyte buffer, elution buffer, etc.). Subsequently,negative pressure may be applied to the gas ports on the other side ofthe chip to load additional fluids (e.g. leading electrolyte buffer).Alternatively, negative pressure may be applied to the gas ports on oneside of the chip to load one or more fluids (e.g. leading electrolytebuffer and trailing electrolyte buffer). Subsequently, negative pressuremay be applied to the gas ports on the other side of the chip to loadadditional fluids (e.g. trailing electrolyte buffer, elution buffer,etc.). Alternatively, negative pressure may be applied to all of the gasports connected to a channel at the same time. The sample may be loadedby applying negative pressure or vacuum before, during, or after loadingof the isotachophoresis buffers. The sample may be loaded withoutapplying negative pressure or vacuum, for example by wetting or gravity.

FIGS. 47A-47B show an exemplary pneumatic control scheme for stagedliquid loading. For each step described in FIG. 47A, a top view of achannel with a plateau capillary barrier 4703 is shown in the center anda side view schematic of the plateau capillary barrier 4703 is shown onthe right. Fluids may be loaded into the reservoirs 4701 on themicrofluidic cartridge and controllably primed to a fixed location andthen merged without creating a bubble as described herein. At Step 1,the two fluids may initially be loaded into reservoirs 4701 connected bya microfluidic channel with a capillary barrier 4703 between them. AtStep 2, the pressure may then be increased and held at fixed pressuresin order to move the menisci 4702 of the two fluids from thereservoir/channel boundary into the channel and up to the base of thecapillary barrier 4703. At Step 3, the pressure may again be increasedincrementally to pull the menisci 4702 to the top of the capillarybarrier 4703. An air gap 4704 may be disposed across the plateau regionof the capillary barrier 4703 in order to maintain a distance betweenthe menisci 4702. At Step 4, the pressure may be increased beyond aholdoff pressure (also referred to herein as a burst pressure) of thebarrier 4703 in order to merge the two fluids and form aliquid-to-liquid interface as described herein. FIG. 47B shows aschematic illustrating Steps 2-4 of the pressure control scheme used tomerge fluids at the capillary barriers 4703 within the microfluidicchannel system. The vacuum pressure may be increased in discrete stepsto move the menisci 4702 of the two fluids to the capillary barrier 4703and then to a connect the fluids as described in FIG. 47A. By increasingthe negative pneumatic pressure in a stepwise manner, the menisci 4702may be merged to form a liquid-to-liquid barrier with little to noactive mixing occurring between the two liquids. This may be of use whenone liquid arrives at the capillary barrier 4703 before the otherliquid. By holding the menisci at opposite ends of the plateau region,the chance of the earlier fluid bursting past the capillary barrier 4703and into the other side of the channel, which may lead to mixing orformation of the liquid-to-liquid interface at an undesirable location,may be greatly reduced compared to a capillary barrier having no plateauregion. As will be understood by one of ordinary skill in the art, thepressure values, increments, and times, can be adjusted to accommodate arange of barrier geometries and fluid wetting properties.

Sensors (e.g., electrodes) can be used to detect liquid filling levelsor bubbles (e.g., via current or voltage sensing) and provide feedback.Geometric features (e.g. constrictions, expansions, or turns) can beused in combination with electrodes to monitor impedance of channels andthereby the time progression of isotachophoresis. For example, duringITP the nucleic acids are focused, and voltage can be used to track thefocused band location in the channel from start to finish. In oneexample, monitoring of fluid expansion into a reservoir (such as anelution reservoir) from a connected channel with smaller cross sectionalarea can be used to determine the time the analyte is eluting, therebyallowing for automated elution and end-process control. In anotherexample, a channel constriction can be designed to allow detection ofthe timing (or triggering) of a step in an electrokinetic process, suchas when the focused analyte is entering a channel zone where a reactionis to take place or where an optical detection event is to take place,allowing control of reaction timing or detector triggering.

For example, FIG. 21 shows one such geometric feature which may be usedfor triggering purposes. An infrared sensor may be positioned at thecenter of a turn in the microfluidic channel in detector location 2105.During isotachophoresis run, this geometric turn feature can facilitatethe generation of a higher local resistance relative to other sectionsof the channel. The high local resistance may be detected in temperatureand/or voltage traces as shown on the left of FIG. 21. For example, anincrease between 2101 and 2012 in the trace of temperature signalcaptured by the infrared sensor during the run may indicate switchingfrom separation to elution process. The two-step temperature risebetween 2103 and 2104 can indicate the geometric turn-enabled featurefor triggering. The arriving time of triggering feature may vary betweenlanes, chips, and instruments. The generic pattern of temperature tracemay persist for the same extraction chemistry. In at least someinstances, such geometric features may provide reduced disturbance fromexternal features.

Reservoir and channel features can be designed to control or preventpressure driven flow. For example, a reservoir (e.g., sample and elutionreservoirs) can have an internal shape designed so that large changes inliquid height produce only small variations in internal volume at theintended head height as shown in FIG. 11. This can provide more precisecontrol of the liquid volume contained in the reservoir. For otherreservoirs, liquid volume can vary without detriment to a separationprocess; such reservoirs can be designed to have large volume changes inresponse to small liquid height changes, and can help stabilize liquidheight throughout the fluidic device. Low fluidic resistances betweenreservoirs can be used to enable fast equilibration times of headpressures and to enable minimal flow of liquids in channels before,during, or after an electrokinetic process.

Reservoirs can be designed to minimize evaporation, for example bycontrolling the surface area within the reservoir to maintain a constantor fixed volume. Reservoirs can be designed to maximize liquid recoveryfrom the reservoirs, for example by using drafted angle wall designs tominimize dead zones. Reservoirs can be designed to prevent the flow ofliquids in connecting channels into the reservoir during unloading,which can help maintain purity or separation of material (e.g., nucleicacids) being unloaded. Reservoirs can be designed for easy loading orunloading via pipetting, for example by having dimensions amenable toadmitting a pipet tip or having volumes within typical pipet operation.For example, the elution reservoir may be configured to admit a pipettip for extraction of nucleic acids. Reservoirs can be designed orspaced to accept multi-channel pipettors (e.g., having a pitch of about9 mm).

Reservoirs (e.g., sample reservoirs) can be located directly above thechannels to be filled, which can minimize liquid lost in connectingchannels between reservoirs and the channels they fill. Reservoirs(e.g., sample reservoirs) can have a conical shaped bottom and acylindrical through-hole or aperture; the large inner diameter at thetop of such a reservoir can allow it to contain a large volume while theliquid meniscus at the bottom of the reservoir has a smaller innerdiameter, reducing the amount of liquid left behind after dispensing.Such a design can also reduce or prevent wicking of wetting fluids intoconcave corners. In some cases, a through-hole from a reservoir (e.g.,sample reservoir) into a channel is less than or equal to about 2millimeters (mm).

FIG. 11 shows a sample reservoir 1100 configured to reduce the amount ofsample left behind (or lost) in the reservoir 1100 after moving thesample to the connected channel 1101. The low-loss sample reservoir 1100may reduce the amount of sample left in the reservoir 1100 after movingthe sample to the connected channel 1101 without adding or pumping inadditional volume (of sample or other fluid) in to the sample reservoir1100 following or during delivery of the sample into the connectedchannel 1101. The low-loss sample reservoir 1100 may comprise an upperor top portion 1102 with an inner hydraulic diameter D₁ configured tocontain a sample volume prior to loading the sample into the channel1101, a lower or bottom portion 1103 with an inner hydraulic diameter orthrough-hole D₂ and height H₁ configured to contain a sample volumeafter loading the sample into the channel 1101, and a tapered or conicalportion 1104 therebetween. In some cases, the upper portion 1102 and/orthe lower portion 1103 are non-symmetrical, in which case the dimensionsD₁ to D₂ may represent the maximum dimension across of the upper and/orlower portions 1102, 1103, respectively.

The sample reservoir 1100 may be configured to produce a head height H₂of sample left behind which equals or nearly equals the head height H₃of the buffers in the other reservoirs 1110 connected to the channel1101 in order to limit, prevent, or reduce pressure-driven flow andmixing in the channel 1101. A standard buffer reservoir 1110 maycomprise an upper portion 1112 with an inner hydraulic diameter D₃ and alower portion 1113 with an inner hydraulic diameter D₄. Unlike in thesample well, D₃ may be substantially similar to D₄ such that a largervolume of fluid is held within the buffer well 1110 compared to thesample well 1100 when the head heights H₂ and H₃ are equal or nearlyequal.

The sample reservoir 1100 may be configured to hold a sample volume(with or without buffer) of at least about 1 nanoliter (nL), 10 nL, 20nL, 50 nL, 100 nL, 200 nL, 500 nL, 1 microliter (μL), 10 μL, 20 μL, 30μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 200 μL, 300 μL,400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, μL, 1 milliliter (mL), 2mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL. In some cases,the sample reservoir 1100 may be configured to hold a sample volumewithin a range of from about 1 nL to about 10 nL.

The inner hydraulic diameter D₁ may be larger than the through-holehydraulic diameter D₂. The inner hydraulic diameter D₁ of the upperportion may be at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm,8 mm, 9 mm, 10 mm, 11 mm, 12, mm 13 mm, 14 mm, or 15 mm. The innerhydraulic diameter D₁ of the upper portion may be at most about 1 mm, 2mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12, mm 13mm, 14 mm, or 15 mm. The inner hydraulic diameter D₂ of the lowerportion may be at least about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm,3.5 mm, 4 mm, 4.5 mm, or 5 mm. The inner hydraulic diameter D₂ of thelower portion may be at most about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

The ratio of D₁ to D₂ may determine the amount of sample left in thesample reservoir after the sample is moved into the channel. In somecases the ratio of D₁ to D₂ is at least about 2:1, 5:1, 10:1, 15:1,20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1. In some cases the ratio orD₁ to D₂ is at most about 2:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1,40:1, 45:1, or 50:1. A ratio of D₁ to D₂ may be greater than 2:1 inorder to facilitate moving at least 50% of the sample volume from thelow-loss sample reservoir 1100 into the channel 1101.

The cross-sectional area of the upper portion may be at least about 3mm², 5 mm², 10 mm², 15 mm², 20 mm², 25 mm², 30 mm², 35 mm², 40 mm², 45mm², 50 mm², 55 mm², 60 mm², 65 mm², 70 mm², 75 mm². The cross-sectionalarea of the upper portion may be at most about 3 mm², 5 mm², 10 mm², 15mm², 20 mm², 25 mm², 30 mm², 35 mm², 40 mm², 45 mm², 50 mm², 55 mm², 60mm², 65 mm², 70 mm², 75 mm². The cross-sectional area of the lowerportion may be at least about 0.2 mm², 0.3 mm², 0.4 mm², 0.5 mm², 1 mm²,1.5 mm², 2 mm², 2.5 mm², 3 mm², 3.5 mm², 4 mm², 4.5 mm², 5 mm², 6 mm², 7mm², 8 mm², 9 mm², 10 mm², 11 mm², 12 mm². The cross-sectional area ofthe lower portion may be at most about 0.2 mm², 0.3 mm², 0.4 mm², 0.5mm², 1 mm², 1.5 mm², 2 mm², 2.5 mm², 3 mm², 3.5 mm², 4 mm², 4.5 mm², 5mm², 6 mm², 7 mm², 8 mm², 9 mm², 10 mm, 11 mm², 12 mm².

The ratio of the cross-sectional area of the upper portion to thecross-sectional area of the lower portion may determine the amount ofsample left in the sample reservoir after the sample is moved into thechannel. In some cases, the ratio for the upper portion cross-sectionalarea to the lower portion cross-sectional area is at least about 4:1,5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1,200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 600:1, 700:1, 800:1,900:1, 1000:1, 1500:1, 2000:1, or 2500:1. In some cases, the ratio forthe upper portion cross-sectional area to the lower portioncross-sectional area is at most about 4:1, 5:1, 10:1, 20:1, 30:1, 40:1,50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1,400:1, 450:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1500:1, 2000:1,or 2500:1.

The tapered portion between the upper portion and the lower portion maycomprise an angle so as to facilitate wetting of sample into the lowerportion and movement of the sample from the low-loss sample well to thechannel. In some cases, the tapered portion of the low-loss samplereservoir may comprise a half-angle between the upper portion and thelower portion of less than about 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°,80°, or 90°. In some cases, the tapered portion of the low-loss samplereservoir may comprise a half-angle between the upper portion and thelower portion of more than about 10°, 20°, 30°, 40°, 45°, 50°, 60°, 70°,80°, or 90°.

In some cases, the height H₁ of the lower portion can be configured soas to produce a head height of sample left behind which equals or nearlyequals the head height of the buffers in the other reservoirs connectedto the channel in order to limit, prevent, or reduce pressure-drivenflow and mixing in the channel. The height H₁ of the lower portion maybe at least about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm,or 10 mm. The height H₂ of the lower portion may be at most about 1 mm,2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm.

Reservoirs (e.g., elution reservoirs) can have diameters that are largecompared to the diffusion length scale of analytes (e.g., nucleic acids)to reduce the diffusion of analytes out of a reservoir. In some cases,the reservoir diameter length scale can be on the order of millimeters,and the resulting diffusion time of analyte from the reservoir can be onthe order of hours. Connections (e.g. through-holes or apertures)between channels and reservoirs (e.g., elution reservoirs) can bedesigned without sharp corners, thereby reducing the prevalence of highelectric field regions at these connections and increasing the residencetime of analytes within the reservoir. In some cases, the cross sectionof a reservoir (e.g., elution reservoir) normal to the electric fieldcan be significantly greater than the cross section of the channelnormal to the electric field, thereby reducing the electric fieldstrength in the reservoir and increasing the residence time of analytewithin the reservoir.

In some cases, an elution channel and/or an elution reservoir cancomprise a second leading electrolyte buffer, different in type orconcentration from the first leading electrolyte buffer used in the mainchannel. This can allow purified material to be eluted in the secondleading electrolyte buffer (e.g., an elution buffer or output solution).The effective mobility magnitude of the second leading electrolyte ionswithin the second leading electrolyte buffer can be greater than theeffective mobility magnitude of the nucleic acids. The second leadingelectrolyte buffer can have low ionic strength, for example an ionicstrength compatible with downstream assays (e.g., qPCR, next generationsequencing). In some cases, the second leading electrolyte buffer is thesame as the first leading electrolyte buffer but present at a differentconcentration or ionic strength (e.g., an ionic strength lower than thatof the first leading electrolyte buffer). For example, the first leadingelectrolyte buffer may have an electrolyte ion concentration of 70-100mM (e.g. 70-100 mM Tris HCl) while the second leading electrolyte buffermay have an electrolyte ion concentration of less than 70 mM, less than60 mM, or less than 50 mM (e.g., less than 50 mM Tris HCl).

Reservoirs (e.g. sample reservoir) can be configured for injection viathe application of pressure or via direct injection. Using a directinjection reservoir, the sample may be injected into the microfluidicchannel via a pipette. The reservoir may be configured such that theoutlet of a pipette tip may be directly coupled to the inlet of thechannel. Proper tip placement in the reservoir may allow the sample tobe injected into the channel while minimizing sample loss to the sidesand/or interior of the reservoir.

FIGS. 48A-48H show exemplary sample inlet reservoir designed for directinjection.

FIGS. 48A-48B show an exemplary design of a sample loading reservoir4800 (or buffer reservoir) designed to promote direct injection of asample (or buffer) into the channel of a microfluidic device. Theloading reservoir 4800 may be configured to guide placement of a pipettetowards a resting place 4801 for the end of the pipette tip by a guidewall 4802. The resting place 4801 is generally an aperture orthrough-hole that penetrates the chip surface. The guide wall 4802 maybe configured to constrain the pipette tip orientation and position toproperly align the pipette tip into the resting place 4801. The guidewall 4802 may, for example, have a frustoconical shape, with thenarrower region of the cone 4804 located at the resting place 4801 andthe wider region of 4803 of the cone located at the entry way forambient air 4807 of the loading reservoir 4800. The wider portion 4803and the narrower portion 4804 may have a circular cross-section. Theguide wall 4802 may taper between the wider portion 4803 and thenarrower portion 4804 at an angle configured to guide the pipette to theresting place 4801. By placing the pipette tip in this location 4801,the user can inject sample directly into the channel by dispensing tothe first stop with a standard laboratory micropipette.

The sample (or buffer) loading reservoir 4800 may be configured tominimize the amount of sample (or buffer) volume that is held in thewell 4800 at a fixed head height by minimizing the diameter of the well4800. The total fluid head height may be within a range of about 1 mm toabout 6 mm from the bottom surface of the channel, for example about 2.8mm from the bottom surface of the channel. The volume of fluid whichremains in the well after loading into the channel, which may becontained between the top of the channel and the sample reservoir 4800(e.g. within the through-hole 4807), may be less than about 15 ul, forexample about 7 ul.

The sample (or buffer) loading reservoir 4800 may be configured to loadthe sample (or buffer) into the channel therebelow under gravitationalforce. The through-hole 4807 may have a diameter within a range of about0.5 mm to about 5 mm, for example about 1.5 mm, for example about 1 mm.

The sample (or buffer) loading reservoir 4800 may comprise afrustoconical shape with a diameter at the fluid head height (e.g. adiameter at the resting place 4801 in the narrower portion 4804) withina range of about 0.1 mm to about 4 mm, for example within a range ofabout 1 mm to about 4 mm, for example about 1.8 mm.

The sample (or buffer) loading reservoir 4800 may have a height of nomore than about 20 mm, no more than about 15 mm, no more than about 10mm, or greater than 10 mm. For example, the sample (or buffer) loadingreservoir 4800 may have a height of about 6 mm. Alternatively, thesample (or buffer) loading reservoir 4800 may have a height within arange of about 8 mm to about 10 mm.

The wider portion 4803 of the sample (or buffer) loading reservoir 4800may have a maximum dimension across of about 4.2 mm.

The narrower portion 4804 of the sample (or buffer) loading reservoir4800 may have a maximum dimension across of about 1.5 mm.

The guide wall 4802 of the sample (or buffer) loading reservoir 4800 maytaper between the wider portion 4803 and the narrower portion 4804 at anangle within a range of about 60 degrees to about 90 degrees.

FIG. 48C shows another exemplary reservoir 4805 geometry which may becompatible with direct injection. Unlike the geometry shown in FIGS.48A-48B, the reservoir 4805 lacks a guide wall to ensure properplacement. The user may manually position their pipette tip to rest onthe resting plane 4806 and then dispense the sample into the channelbelow via the aperture 4807 in the resting plan 4806. The reservoir 4805may for example have an elongate cross-sectional shape configured tocapture overflow liquid from the pipette during injection and ensurebubble-free loading of the entire liquid volume. FIG. 48D shows theresult of using the reservoir 4805 in FIG. 48A to inject a fluorescentaqueous solution 4808 into a microfluidic channel. Liquid 4808 wasinjected via the aperture 4807 in the guide plane 4806 of the reservoir4805 and was able to fill the adjoining channel without bubbleformation.

The sample (or buffer) loading reservoir 4805 may be configured tominimize the amount of sample (or buffer) volume that is held in thewell 4805 at a fixed head height by minimizing the diameter of the well4805. The total fluid head height may be within a range of about 1 mm toabout 6 mm from the bottom surface of the channel, for example about 2.8mm from the bottom surface of the channel. The volume of fluid whichremains in the well after loading into the channel, which may becontained between the top of the channel and the sample reservoir 4805(e.g., within the through-hole 4807), may be less than about 15 ul, forexample about 7 ul.

The sample (or buffer) loading reservoir 4805 may be configured to loadthe sample (or buffer) into the channel therebelow under gravitationalforce. The through-hole 4807 may have a diameter within a range of about0.5 mm to about 5 mm, for example about 1.5 mm, for example about 1 mm.

The sample (or buffer) loading reservoir 4805 may comprise an elongateshape, for example an elliptical shape, having a maximum diameter acrossof about 10 mm and a minimum diameter across of about 3 mm.

The sample (or buffer) loading reservoir 4805 may have a height of nomore than about 20 mm, no more than about 15 mm, no more than about 10mm, or greater than 10 mm. For example, the sample (or buffer) loadingreservoir 4805 may have a height of about 6 mm. Alternatively, thesample (or buffer) loading reservoir 4805 may have a height within arange of about 8 mm to about 10 mm.

FIGS. 48E-48F shows an exemplary design of a sample loading reservoir4810 (or buffer reservoir) which may facilitate direct injection of asample (or buffer) into the channel of a microfluidic device. Theloading reservoir 4810 may be substantially similar to the reservoir4800 of FIGS. 48A-48B except that the resting place 4811 form may be asquare aperture 4813 instead of a circular aperture. The loadingreservoir 4810 may be configured to guide placement of a pipette towardsa resting place 4811 for the end of the pipette tip by a guide wall4812. The guide wall 4812 may be configured to constrain the pipette tiporientation and position to properly align the pipette tip into theresting place 4811. The guide wall 4812 may for example have afrustoconical shape, with a narrower region 4814 of the cone located atthe resting place 4811 and the wider region 8413 of the cone located atthe entryway for ambient air 4817 of the loading reservoir 4810. Thewider portion 4813 and the narrower portion 4814 may have a circularcross-section. The resting place 4811 may have a square cross-section atthe base of the guide wall 4812. The guide wall 4812 may taper betweenthe wider portion 4813 and the narrower portion 4814 at an angleconfigured to guide the pipette to the resting place 4811. By placingthe pipette tip in this location 4811, the user can inject sampledirectly into the channel by dispensing to the first stop with astandard laboratory micropipette.

The sample (or buffer) loading reservoir 4810 may be configured tominimize the amount of sample (or buffer) volume that is held in thewell 4810 at a fixed head height by minimizing the diameter of the well4810. The total fluid head height may be within a range of about 1 mm toabout 6 mm from the bottom surface of the channel, for example about 2.8mm from the bottom surface of the channel. The volume of fluid whichremains in the well after loading into the channel, which may becontained between the top of the channel and the sample reservoir 4810(e.g., within the through-hole 4817), may be less than about 15 ul, forexample about 7 ul.

The sample (or buffer) loading reservoir 4810 may be configured to loadthe sample (or buffer) into the channel therebelow under gravitationalforce. The through-hole 4817 may have a diameter within a range of about0.5 mm to about 5 mm, for example about 1.5 mm, for example about 1 mm.

The sample (or buffer) loading reservoir 4810 may have a height of nomore than about 20 mm, no more than about 15 mm, no more than about 10mm, or greater than 10 mm. For example, the sample (or buffer) loadingreservoir 4810 may have a height of about 6 mm. Alternatively, thesample (or buffer) loading reservoir 4810 may have a height within arange of about 8 mm to about 10 mm.

The wider portion 4813 of the sample (or buffer) loading reservoir 4810may have a maximum dimension across of about 2.1 mm.

The narrower portion 4814 of the sample (or buffer) loading reservoir4810 may have a maximum dimension across of about 1.5 mm.

The guide wall 4812 of the sample (or buffer) loading reservoir 4810 maytaper between the wider portion 4813 and the narrower portion 4814 at anangle within a range of about 60 degrees to about 90 degrees.

The sample (or buffer) reservoir 4810 can have one or more through-holes4817 having a shape configured to allow some portion of a currentpassing through the sample channel bellow the reservoir 4810 to enterthe sample reservoir 4810, and to carry analyte out of the reservoir4810 and into the channel below. The through holes 4817 can have adimension substantially or entirely co-extensive with the width of thechannel over which the reservoir 4817 is positioned. This dimension ofco-extension can be, e.g., at least 50% to 150% the width of thechannel, e.g, between about 1 mm and about 5 mm. For example,through-hole 4817 can take a rectangular shape, including a square. Thethrough-hole 4817 also can assume a rounded shape, including a circle oran oval.

The rectangular aperture 4813 may be large enough to allow some portionof the electric field applied to the fluidic circuit to interact withthe target nucleic acids of the sample. For example, if a portion of thesample remains in the reservoir 4810 after loading, when the electricfield is applied, the electric field may induce migration of theremaining sample nucleic acids from the reservoir 4810 into the channelfor ITP concentration. Thus, even if some of the sample buffer volumeremains in the well 4810 after loading, the target nucleic acids maystill enter the channel and less of the analyte may be lost to the well4810.

When an electric field is applied to the ITP branch, greater than 10% ofan electric current applied may travel above a top surface of saidsample channel across a length of said sample reservoir.

The rectangular aperture 4813 may have a width within a range of about80% to about 120% of a width of said sample channel, for example about100% of the sample channel (e.g. a width of 2.2 mm for a 2.2 mmchannel).

FIGS. 48G-48H shows an exemplary design of a sample loading reservoir4820 (or buffer reservoir) which may facilitate direct injection of asample (or buffer) into the channel of a microfluidic device. The sampleloading reservoir 4820 may comprise an oval-shaped reservoir having twosample injection ports 4821 through which the user can inject sampleinto the channel. The reservoir 4810 may comprise a guide wall 4822configured to guide placement of a pipette towards one or both of thesample injection ports 4821. The guide wall 4822 may for example have anarrower region 4814 of the cone located at the resting place 4811 andthe wider region 8413 of the cone located at the entryway for ambientair 4817 of the loading reservoir 4810. The wider portion 4823 and thenarrower portion 4824 may have an oval or elongate cross-section. Theguide wall 4822 may taper between the wider portion 4823 and thenarrower portion 4824 at an angle configured to guide the pipette to oneor both of the sample injection ports 4821. By placing the pipette tipin either port 4821, the user can inject sample directly into thechannel by dispensing to the first stop with a standard laboratorymicropipette.

The sample (or buffer) loading reservoir 4820 may be configured tominimize the amount of sample (or buffer) volume that is held in thewell 4820 at a fixed head height by minimizing the diameter of the well4820. The total fluid head height may be within a range of about 1 mm toabout 6 mm from the bottom surface of the channel, for example about 2.8mm from the bottom surface of the channel. The volume of fluid whichremains in the well after loading into the channel, which may becontained between the top of the channel and the sample reservoir 4820(e.g., within the through-holes 4821), may be less than about 15 ul, forexample about 7 ul.

The sample (or buffer) loading reservoir 4820 may be configured to loadthe sample (or buffer) into the channel therebelow under gravitationalforce. The through-holes 4821 may have a diameter within a range ofabout 0.5 mm to about 5 mm, for example about 1.5 mm, for example about1 mm.

The through-holes 4821 may be large enough to allow some portion of theelectric field applied to the fluidic circuit to interact with thetarget nucleic acids of the sample. For example, if a portion of thesample remains in the reservoir 4820 after loading, when the electricfield is applied, the electric field may induce migration of theremaining sample nucleic acids from the reservoir 4820 into the channelfor ITP concentration. Thus, even if some of the sample buffer volumeremains in the well 4820 after loading, the target nucleic acids maystill enter the channel and less of the analyte may be lost to the well4820.

The reservoir 4820 may be oval-shaped to provide a path for the electricfield.

The through-holes 4821 may be separated by a bar or filler block 4825.The bar 4825 across the bottom of the well may 4820 help direct theelectric field into the fluid held in the reservoir 4820 (above thechannel). The bar 4825 may also help stabilize the fluid in the well4820 against secondary flows due to buoyant effects as described herein.

When an electric field is applied to the ITP branch, greater than 10% ofan electric current applied may travel above a top surface of saidsample channel across a length of said sample reservoir 4820.

The through-holes 4821 may have a width within a range of about 80% toabout 120% of a width of said sample channel, for example about 100% ofthe sample channel (e.g. a width of 2.2 mm for a 2.2 mm channel).

The through-holes 4821 may have areas within a range of about 0.2 mm² toabout 7 mm², for example within a range of about 0.8 mm² to about 1.5mm² or within a range of about 1 mm² to about 2.75 mm².

The through-holes 4821 may have substantially the same shape. Thethrough-holes 4821 may have different shapes.

The filler block or bar 4825 may have a width of about 3.7 mm spanningthe well 4820 along a lateral axis of the well 4820. The filler block orbar 4825 may have a width of about 2 mm spanning the well 4820 along alongitudinal axis of the well 4820.

The sample reservoir 4820 may comprise a filler block 4825 between 0.2mm and 2 mm in height in the channel, for example about 1.2 mm. Thefiller block 4825 may bifurcate the electric current into upper andlower branches.

The sample (or buffer) loading reservoir 4820 may comprise an elongateshape, for example an elliptical shape, having a maximum diameter (i.e.length) across of about 7.5 mm and a minimum diameter across (i.e.width) of about 5 mm.

The sample (or buffer) loading reservoir 4820 may have a height of nomore than about 20 mm, no more than about 15 mm, no more than about 10mm, or greater than 10 mm. For example, the sample (or buffer) loadingreservoir 4820 may have a height of about 6 mm. Alternatively, thesample (or buffer) loading reservoir 4820 may have a height within arange of about 8 mm to about 10 mm.

The wider portion 4823 of the sample (or buffer) loading reservoir 4820may have a maximum dimension across of about 2.1 mm.

The narrower portion 4824 of the sample (or buffer) loading reservoir4820 may have a maximum dimension across of about 1.5 mm.

The guide wall 4822 of the sample (or buffer) loading reservoir 4820 maytaper between the wider portion 4823 and the narrower portion 4824 at anangle within a range of about 60 degrees to about 90 degrees.

FIGS. 49A and 49B show an exemplary elution channel configured forreducing dispersion while retaining high fluidic and electricalresistance for automated sensing. FIG. 49A shows the channel design. Thechannel may begin as a wide channel 4900 before narrowing 4902. Theaxial distance along which the channel narrows may be between 2 and 50times the maximum channel width. The channel may narrow to a minimumwidth before completing a 90 degree turn 4901. This slow transition intoa narrow passage may generate a low-dispersion turn which can retain thenucleic acid sample in a tight band while turning. As described herein,the detection point for temperature, conductivity, and/or voltagemeasurements for automated signal processing may be located at thecenter of the turn 4901, which may also be the narrowest point of thechannel. This detection point may be located a short distance from theelution reservoir 4903, thereby allowing signal processing decisions(e.g. triggering) to be made in advance of DNA elution. FIG. 49B showsan example of data collected from the channel in FIG. 49A. The x-axis isthe time since the beginning of the separation. The y-axis is thetemperature of the detection point 4901. At time 4904, the ion in frontof the detector is replaced by a new, lower-mobility ion (e.g. leadingelectrolyte) via isotachophoresis. At time 4905, a third ion (e.g. ITPband) displaces the second, resulting in a second temperature rise. Attime 4906, the automated signal processing system detects this changeand turns off the voltage (and current), causing a rapid decrease intemperature within the turn 4901.

The elution reservoir can be configured to collect analyte duringisotachophoresis. For example, the elution chamber can be positionedover a channel which comprises a break. Through-holes from the breakpoints into the reservoir will require current to flow through thechannel, into the reservoir through a first through-hole, and out asecond through-hole into the continuation of the channel. That is, thefluidic circuit is designed so that current cannot flow directly underthe elution reservoir, but must pass into and out of the reservoir. Sucha configuration is shown in FIG. 50A1, which shows a fluidic surface5050 of a substrate with first channel 5051 terminating in through-holes5052, 5053 that communicate with a reservoir on an opposing, reservoirsurface of the substrate. (See. e.g., FIGS. 51C and 51D.) In someembodiments, the through-hole 5052 entering the reservoir in thedirection of analyte flow from channel 5051 is positioned lower in thereservoir than the through hole 5053 leaving the reservoir towardssecond channel 5054. This creates a volume in the reservoir between thethrough holes 5052, 5053 which must be filled to create an electricalpath into and out of the reservoir. The first channel 5051 maycommunicate with a first electrode and the second channel 5054 maycommunicate with a second electrode. The channels 5051, 5054 andreservoir may comprise an electrically conductive fluid (e.g. elutionbuffer and/or high concentration elution buffer). Application of avoltage across the first and second electrodes may produce a currentthat travels through the reservoir. Analyte moving into the reservoirtends to remain in the reservoir rather than moving in an upward andoutward direction via channel 5054 toward additional reservoir(s) 5055(e.g. elution buffering reservoir). The reservoir can include a step orplatform positioned, e.g., about 0.5 mm to about 3 mm above the floor ofthe reservoir (e.g., as shown in FIG. 52B). The second through-hole 5053can penetrate this step. Such a step or ramp also creates a well-shapedarea adapted for pipetting.

In some instances, there may be two steps within the reservoir betweenthe first and second through-holes 5052, 5053. The second through-hole5053 may penetrate the higher step which may be further away from thefirst through-hole 5052 than the lower step. Alternatively, the secondthrough-hole 5053 may penetrate the lower step which may be closer thefirst through-hole 5052 than the higher step.

FIG. 50A is a technical drawing top view of an exemplary elutionreservoir 5000 which may be located on any of the chip devices describedherein. The reservoir 5000 may be configured to hold a volume of elutionbuffer as described herein. Upon completion of an ITP run, the targetnucleic acids may reside within this volume as described herein. Thereservoir 5000 may comprise a first through-hole or aperture 5001 and asecond through-hole or aperture 5002. The first and second through-holes5001, 5002 may penetrate the chip surface and be in communication with afluidic channel 5003 located below the reservoir 5000 as describedherein. The fluidic channel 5003 may for example be an elution channelas described herein. The first and second through-holes 5001, 5002 maybe generally aligned with each other and with a longitudinal axis of thefluidic channel 5003 such that application of a fluid into the channel5003 via the second through-hole 5002 pushes the fluid along the channel5003 towards the first through-hole 5001. A user may for example removea first volume out of the first through-hole 5001, for example using apipet. Subsequently, fresh elution buffer may be added to the channel5003 via the second through-hole 5002, for example using a pipet. Theuser may then perform a final evacuation of all remaining buffer fromthe first through-hole 5001, for example using a pipet. In this way, theuser may improve the overall yield of the ITP purification process andensure that the majority of the purified target nucleic acids may beretrieved from the device in two elution steps.

The first through-hole 5001 may for example have an elliptical shape.Alternatively, the first through-hole 5001 may be circular. The firstthrough-hole 5001 may have any shape desired by one or ordinary skill inthe art.

The second through-hole 5002 may for example be circular. Alternatively,the second through-hole 5002 may have an elliptical shape. The secondthrough-hole 5002 may have any shape desired by one or ordinary skill inthe art.

The first through-hole 5001 may have a diameter within a range of about100 um to about 3 mm.

The first through-hole 5001 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

The second through-hole 5002 may have a diameter within a range of about100 um to about 3 mm.

The second through-hole 5002 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

In some embodiments, the channel 5003 below the reservoir 5000 maycomprise a break as shown in FIG. 50A1 and the first and secondthrough-holes 5001, 5002 may be in fluidic and/or electricalcommunication with first and second terminating ends of the channel 5003as described herein. The first and second through-holes 5001, 5002 maybe positioned at different heights within the reservoir. The firstthrough-hole 5001 may enter the reservoir 5000 at a position lower inthe reservoir than the second through-hole 5002. The first and secondthrough-holes 5001, 5002 may be separated by a step or platform asdescribed herein.

The reservoir 5000 may be circular. The reservoir 5000 may have adiameter of about 5.3 mm.

The reservoir 5000 may be defined by a wall having a height of no morethan 25 mm, no more than 15 mm, no more than 10 mm, or greater than 10mm. The reservoir 5000 may have height of about 6 mm. The reservoir 5000may have height within a range of about 8 mm to about 10 mm.

The first and second through holes 5001, 5002 may have areas within arange of about 0.2 mm² to 7 mm2. The first through-hole 5001 may have anarea within a range of about 1 mm² to about 2.75 mm². For example thesecond through-hole 5002 may have an area within a range of about 0.8mm² to about 1.5 mm².

The through-holes 5001, 5002 may have substantially the same shape. Thethrough-holes 5001, 5002 may have different shapes.

The first through-hole 5001 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel 5003 or the width of the reservoir 5000.

The second through-hole 5002 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel 5003 or the width of the reservoir 5000.

The through-holes 5001, 5002 may be arranged along a longitudinal axisof the channel 5003.

The first through-hole 5001 may have a maximum distance across (i.e.length) of about 1.75 mm and a minimum distance across (i.e. width) ofabout 1.3 mm.

The second through-hole 5002 may be relatively circular with a diameterof about 1.2 mm. The second through-hole 5002 may be shaped so as tospread the electrical field evenly across the reservoir 5000.

The volume of the reservoir 5000 between the first and secondthrough-holes 5001, 5002 may be no more than about 2.5 ml, 1 ml, or 0.5ml. The volume of the reservoir 5000 between the first and secondthrough-holes 5001, 5002 may be about 0.1 mL.

At least part of each through-hole 5001, 5002 may be substantiallyco-extensive with the fluidic channel 5003 across said width of saidfluidic channel 5003.

The first through-hole 5001 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel 5003 and/ora width of the reservoir 5000. The at least one side(s) may (each) be atleast 75% linear.

The second through-hole 5002 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel 5003 and/ora width of the reservoir 5000. The at least one side(s) may (each) be atleast 75% linear.

The first and second through-holes 5001, 5002 may have a shape that,when said fluidic channel 5003 and said reservoir 5000 comprise anelectrically conductive fluid and an electric current is passed throughsaid fluidic channel 5003, at least 5%, at least 6%, at least 7%, atleast 10%, or at least 20% of said electric current passes through saidreservoir 5000 between the first and the second through-holes 5001,5002.

FIG. 50B shows a cross-sectional view of another exemplary elutionreservoir 5010 which may be located on any of the chip devices describedherein. FIG. 50B shows another view of the elution reservoir design 603shown in FIG. 6A. The reservoir 5010 may be configured to hold a volumeof elution buffer as described herein. Upon completion of an ITP run,the target nucleic acids may reside within this volume as describedherein. The reservoir 5010 may comprise a single through-hole oraperture 5011. The through-hole 5011 may penetrate the chip surface andbe in communication with a fluidic channel located below the reservoir5010 as described herein. The fluidic channel may for example be anelution channel as described herein. A user may, for example, remove afirst volume of fluid out of the reservoir 5010 via the through-hole5011, for example using a pipet. Subsequently, fresh elution buffer maybe added to the channel via the through-hole 5011 of reservoir 5010, forexample using a pipet. The user may then perform a final evacuation ofall remaining buffer from the through-hole 5011, for example using apipet. In this way, the user may improve the overall yield of the ITPpurification process and ensure that the majority of the purified targetnucleic acids may be retrieved from the device in two elution steps.

FIG. 50C-50E are background subtracted fluorescence images of a fluidicdevice at different steps of elution for reservoir design 5000 in FIG.50A. FIG. 50C represents the state of the chip immediately after thecompletion of an ITP run, with successful channel closure to preventfluid movement within the elution reservoir 5000 and channels (e.g.channel 5003) as described herein. FIG. 50D shows the same chip, afterthe initial elution from the first through-hole 5001 as describedherein. FIG. 50E is the final state of the chip, after the user hasadded additional elution buffer to the second through-hole 5002, andfully evacuated the elution reservoir 5000 via pipetting from the firstthrough-hole 5001. As is evident by these background-subtracted images,the majority of the DNA material may be obtained from the first elution,but the second elution step may enable the user to wash and retrieve theremaining DNA from the reservoir. FIG. 50F is a block diagram showingthe steps in the elution workflow for reservoir 5000. At Step 5020, ITPmay be completed as described herein. At Step 5021, the elution channelmay be closed as described herein. At Step 5022, a first elution may beperformed by removing the eluate from the first through-hole 5001 of theelution reservoir 5000. At Step 5023, additional elution buffer may beadded to the second through-hole 5002 of the elution reservoir 5000. AtStep 5024, a second elution may be performed by removing the eluate fromthe first through-hole 5001 of the elution reservoir 5000.

Provided herein are elution methods or processes that can be used withmore than one elution reservoir design after completing an ITP run (Step5020). The instrument channel closer can isolate the DNA in astabilizing buffer (elution buffer) (Step 5021). The elution strategydescribed herein is generally designed to take advantage of thisisolation, and maximize the total DNA yield. The first step of theelution technique may be to remove the majority of the liquid volume(i.e. elution buffer plus nucleic acid that has made it to thereservoir) in the reservoir 5000 (or any elution reservoir describedherein). This may be done by placing a pipette at the first reservoirthrough-hole 5001 and aspirating between 30 and 90 uL of fluid therefrom(Step 5022). Secondly, the user may wash the reservoir 5000 using cleanbuffer (Step 5023). The user may wash from the second through-hole 5002toward the first through-hole 5001 in an attempt to drive the maximumamount of analyte to the aspiration point of the first through-hole5001. The user may, for example, accomplish this by pipetting 10 to 50uL of fresh buffer into the second through-hole 5002 of the elutionreservoir 5000. Finally, the user may recover the remaining analyte byaspirating as much volume as possible from the first through-hole 5001(Step 5024). This multi-step elution workflow can allow the user toobtain higher or the maximum possible DNA yield.

The final stage of nucleic acid (NA) processing in the chip device maygenerally be to transfer the nucleic acids from a microfluidic channelinto a reservoir (elution reservoir). This reservoir may be accessiblevia a pipette, unlike the channel. This may allow a user to recover thematerial (nucleic acid) following completion of the ITP run and anysubsequent on-chip processing as described herein.

Decreasing the volume in this reservoir may be beneficial because it mayincrease the concentration of the recovered nucleic acid. Providedherein is a reservoir designed with low liquid volume (˜50 uL), butwhich takes a long time for nucleic acid to traverse.

FIG. 51A shows an exemplary fluidic reservoir 5100 which can be used fornucleic acid elution. The reservoir 5100 may be located on any of thechip devices described herein. The reservoir 5100 may have a circularcross-section. The reservoir 5100 may be configured to hold a volume ofelution buffer as described herein. Upon completion of an ITP run, thetarget nucleic acids may reside within this volume as described herein.The reservoir 5100 may comprise a first through-hole or aperture 5101and a second through-hole or aperture 5102. Each of the twothrough-holes 5101, 5102 may be connected to a fluidic channel 5104 on abottom layer of the fluidic device. The first and second through-holes5101, 5102 may penetrate the chip surface and be in communication with afluidic channel 5104 located below the reservoir 5100 as describedherein. The fluidic channel 5104 may for example be an elution channelas described herein. The first through-hole 5101 may include a steeprise in fluid height (e.g. from 100-400 um at the channel to 2-3 mmwithin the well) from the channel 5104 to a plateau 5103 on which thetarget nucleic acid may be captured prior to elution. Another verticalrise 5106 within the reservoir 5100 may couple the plateau 5103 to thesecond through-hole 5102. The first through-hole 5101 and the secondthrough-hole 5102 may be one different vertical planes within thereservoir 5100. The first and second through-holes 5101, 5102 may begenerally aligned with each other and with a longitudinal axis of thefluidic channel 5104 such that application of a fluid into the channel5104 via the second through-hole 5102 pushes the fluid along the channel5104 towards the first through-hole 5101 as described herein. The secondthrough-hole 5102 may be used to create a fluidic connection andelectrical connection to a high-voltage electrode. FIG. 51B showsanother embodiment of the reservoir 5105, which may be substantiallysimilar to reservoir 5100 with further changes for injection moldingcompatibility. For example, the second through-hole step 5106 may beminimized in size except where needed for the second through-hole wall.

The first through-hole 5101 may for example have an elliptical shape.Alternatively, the first through-hole 5101 may be circular. The firstthrough-hole 5101 may have any shape desired by one or ordinary skill inthe art.

The second through-hole 5102 may for example be circular. Alternatively,the second through-hole 5102 may have an elliptical shape. The secondthrough-hole 5002 may have any shape desired by one or ordinary skill inthe art.

The first through-hole 5101 may have a diameter within a range of about100 um to about 3 mm.

The first through-hole 5101 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

The second through-hole 5102 may have a diameter within a range of about100 um to about 3 mm.

The second through-hole 5102 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

The first through-hole 5101 may be an elongate through-hole thatpenetrates the surface of the chip to couple the reservoir 5100 to thechannel 5104. The first through-hole 5101 may, for example, becylindrical as described herein. The first through-hole 5101 may, forexample, have a height from the channel 5104 to the reservoir 5100within a range of about 1 mm to about 3 mm.

The second through-hole 5102 may be an elongate through-hole thatpenetrates the surface of the chip to couple the reservoir 5100 to thechannel 5104. The second through-hole 5102 may, for example, becylindrical as described herein.

The second through-hole 5102 may have a height above the channel 5104greater than the height above the channel of the first through-hole5101.

The reservoir 5100 may comprise a circular cross-section. Alternatively,the reservoir 5100 may comprise an elongate or oval cross-section.

In some embodiments, the channel 5104 below the reservoir 5100 maycomprise a break as shown in FIG. 50A1 and the first and secondthrough-holes 5101, 5102 may be in fluidic and/or electricalcommunication with first and second terminating ends of the channel 5104as described herein. The first and second through-holes 5101, 5102 maybe positioned at different heights within the reservoir. The firstthrough-hole 5101 may enter the reservoir 5100 at a position lower inthe reservoir than the second through-hole 5102. The first and secondthrough-holes 5101, 5102 may be separated by a step or platform 5106 asdescribed herein.

The reservoir 5100 may be circular. The reservoir 5100 may have adiameter of about 5.3 mm.

The reservoir 5100 may be defined by a wall having a height of no morethan 25 mm, no more than 15 mm, no more than 10 mm, or greater than 10mm. The reservoir 5100 may have height of about 6 mm. The reservoir 5100may have height within a range of about 8 mm to about 10 mm.

The first and second through holes 5101, 5102 may have areas within arange of about 0.2 mm² to 7 mm2. The first through-hole 5101 may have anarea within a range of about 1 mm² to about 2.75 mm². For example thesecond through-hole 5102 may have an area within a range of about 0.8mm² to about 1.5 mm².

The through-holes 5101, 5102 may have substantially the same shape. Thethrough-holes 5101, 5102 may have different shapes.

The first through-hole 5101 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel 5104 or the width of the reservoir 5100.

The second through-hole 5102 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel 5104 or the width of the reservoir 5100.

The through-holes 5101, 5102 may be arranged along a longitudinal axisof the channel 5003.

The first through-hole 5101 may have a maximum distance across (i.e.length) of about 1.75 mm and a minimum distance across (i.e. width) ofabout 1.3 mm.

The second through-hole 5102 may be relatively circular with a diameterof about 1.2 mm. The second through-hole 5102 may be shaped so as tospread the electrical field evenly across the reservoir 5100.

The second through-hole 5102 may enter the reservoir though a platform5106 in the reservoir 5100 positioned about 1 mm to about 6 mm above apoint of entry into the reservoir 5100 of the first through-hole 5101.

The volume of the reservoir 5100 between the first and secondthrough-holes 5101, 5102 may be no more than about 2.5 ml, 1 ml, or 0.5ml. The volume of the reservoir 5100 between the first and secondthrough-holes 5101, 5102 may be about 0.1 mL.

At least part of each through-hole 5101, 5102 may be substantiallyco-extensive with the fluidic channel 5104 across said width of saidfluidic channel 5104.

The first through-hole 5101 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel 5104 and/ora width of the reservoir 5100. The at least one side(s) may (each) be atleast 75% linear.

The second through-hole 5102 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel 5104 and/ora width of the reservoir 5100. The at least one side(s) may (each) be atleast 75% linear.

The first and second through-holes 5101, 5102 may have a shape that,when said fluidic channel 5104 and said reservoir 5100 comprise anelectrically conductive fluid and an electric current is passed throughsaid fluidic channel 5104, at least 5%, at least 6%, at least 7%, atleast 10%, or at least 20% of said electric current passes through saidreservoir 5100 between the first and the second through-holes 5101,5102.

FIGS. 51C and 51D show two views of another embodiment of a reservoir5110. The reservoir 5110 may be located on any of the chip devicesdescribed herein. The reservoir 5110 may have an elongate or ovalcross-section. The reservoir 5110 may be configured to hold a volume ofelution buffer as described herein. Upon completion of an ITP run, thetarget nucleic acids may reside within this volume as described herein.The reservoir 5110 may comprise a first through-hole or aperture 5111and a second through-hole or aperture 5112. Each of the twothrough-holes 5111, 5112 may be connected to a fluidic channel on abottom layer of the fluidic device. The first and second through-holes5111, 5112 may penetrate the chip surface and be in communication with afluidic channel located below the reservoir 5110 as described herein.The fluidic channel may for example be an elution channel as describedherein. The first through-hole 5111 and the second through-hole 5111 maybe on the same plane within the reservoir 5110. The first through-hole5111 and the second through-hole 5111 may be separated by vertical gates5113 that rise above the plane of the through-holes 5111, 5112. Thevertical gates 5113 may act to drive the target nucleic acids up intothe reservoir 5110. This may facilitate the buoyant effect describedherein and aid in lifting the target nucleic acids from the channel intothe reservoir 5110. In some instances, ITP may be performed at a lowerdriving current than necessary to lift the target nucleic acids into thereservoir by the buoyant effect alone (without the aid of the verticalgates 5113). The first and second through-holes 5111, 5112 may begenerally aligned with each other and with a longitudinal axis of thefluidic channel such that application of a fluid into the channel viathe second through-hole 5112 pushes the fluid along the channel towardsthe first through-hole 5111 as described herein. The second through-hole5112 may be used to create a fluidic connection and electricalconnection to a high-voltage electrode.

The first through-hole 5111 may for example have a D-shapedcross-section with a straight wall defied by one of the vertical gates5113. Alternatively, the first through-hole 5111 may have an ellipticalshape. Alternatively, the first through-hole 5111 may be circular. Thefirst through-hole 5111 may have any shape desired by one or ordinaryskill in the art.

The second through-hole 5112 may for example have a D-shapedcross-section with a straight wall defied by one of the vertical gates5113. Alternatively, the second through-hole 5112 may have an ellipticalshape Alternatively, the second through-hole 5112 may be circular. Thesecond through-hole 5112 may have any shape desired by one or ordinaryskill in the art.

The first through-hole 5111 may have a diameter within a range of about100 um to about 3 mm. The first through-hole 5111 may have a diameter ofabout 1.2 mm.

The first through-hole 5111 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

The second through-hole 5112 may have a diameter within a range of about100 um to about 3 mm. The first through-hole 5111 may have a diameter ofabout 1.2 mm.

The second through-hole 5112 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

The first through-hole 5111 may be an elongate through-hole thatpenetrates the surface of the chip to couple the reservoir 5110 to thechannel. The first through-hole 5111 may, for example, be cylindrical asdescribed herein. The first through-hole 5111 may, for example, have aheight from the channel to the reservoir 5110 within a range of about 1mm to about 3 mm.

The second through-hole 5112 may be an elongate through-hole thatpenetrates the surface of the chip to couple the reservoir 5110 to thechannel. The second through-hole 5112 may, for example, be cylindricalas described herein.

The vertical gate 5113 may have a height above the channel greater thanthe height above the channel of the first through-hole 5111.

The vertical gate 5113 may have a height above the channel greater thanthe height above the channel of the second through-hole 5112.

The reservoir 5110 may comprise a circular cross-section. Alternatively,the reservoir 5110 may comprise an elongate or oval cross-section.

The reservoir 5110 may comprise an elongate shape, for example anelliptical shape, having a maximum diameter (i.e. length) across ofabout 7 mm and a minimum diameter across (i.e. width) of about 3.4 mm.

The reservoir 5110 may have a height of no more than about 20 mm, nomore than about 15 mm, no more than about 10 mm, or greater than 10 mm.For example, reservoir 5110 may have a height of about 6 mm.Alternatively, the reservoir 5110 may have a height within a range ofabout 8 mm to about 10 mm.

In some embodiments, the channel below the reservoir 5110 may comprise abreak as shown in FIG. 50A1 and the first and second through-holes 5111,5112 may be in fluidic and/or electrical communication with first andsecond terminating ends of the channel as described herein. The firstand second through-holes 5111, 5112 may be positioned at differentheights within the reservoir 5110. The first through-hole 5111 may enterthe reservoir 5110 at a position lower in the reservoir than the secondthrough-hole 5112. The first and second through-holes 5111, 512 may beseparated steps or gates 5113 as described herein.

The reservoir 5110 may be defined by a wall having a height of no morethan 25 mm, no more than 15 mm, no more than 10 mm, or greater than 10mm. The reservoir 5110 may have height of about 6 mm. The reservoir 5110may have height within a range of about 8 mm to about 10 mm.

The first and second through holes 5111, 5112 may have areas within arange of about 0.2 mm² to 7 mm2. The first through-hole 5111 may have anarea within a range of about 1 mm² to about 2.75 mm². For example thesecond through-hole 5102 may have an area within a range of about 0.8mm² to about 1.5 mm².

The through-holes 5111, 5112 may have substantially the same shape. Thethrough-holes 5101, 5102 may have different shapes.

The first through-hole 5111 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel or the width of the reservoir 5110.

The second through-hole 5112 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel or the width of the reservoir 5110.

The through-holes 5111, 5112 may be arranged along a longitudinal axisof the channel.

The first through-hole 5111 may have a maximum distance across (i.e.length) of about 1.2 mm.

The second through-hole 5112 may have a maximum distance across (i.e.length) of about 1.2 mm.

The volume of the reservoir 5110 between the first and secondthrough-holes 5111, 5112 may be no more than about 2.5 ml, 1 ml, or 0.5ml. The volume of the reservoir 5110 between the first and secondthrough-holes 5111, 5112 may be about 0.1 mL.

At least part of each through-hole 5111, 5112 may be substantiallyco-extensive with the fluidic channel across said width of said fluidicchannel.

The first through-hole 5111 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel and/or awidth of the reservoir 5110. The at least one side(s) may (each) be atleast 75% linear.

The second through-hole 5112 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel and/or awidth of the reservoir 5110. The at least one side(s) may (each) be atleast 75% linear.

The first and second through-holes 5111, 5112 may have a shape that,when said fluidic channel and said reservoir 5110 comprise anelectrically conductive fluid and an electric current is passed throughsaid fluidic channel, at least 5%, at least 6%, at least 7%, at least10%, or at least 20% of said electric current passes through saidreservoir 5110 between the first and the second through-holes 5111,5112.

FIG. 51E shows a comparison of the residence time of nucleic acidbetween the reservoir design 5110 and a reference design 5150. Theresidence time is scaled by applied current and the liquid volume insidethe reservoir to produce a volumetric residence charge, with units C/uL.There was a greater than four-fold increase in volumetric residencecharge from the gated design 5110 (n=2) compared to the reference design5150 without a gate (n=8). FIG. 51F shows a cross-section of thereference design 5150 having a drafted vertical cylinder without anyinternal structure. The reference design 5150 was substantially similarto the reservoir described in FIG. 50B.

FIG. 52A shows another embodiment of a fluidic reservoir 5200. Thereservoir 5200 may be located on any of the chip devices describedherein. The reservoir 5200 may have a circular cross-section. Thereservoir 5200 may be configured to hold a volume of elution buffer asdescribed herein. Upon completion of an ITP run, the target nucleicacids may reside within this volume as described herein. The reservoir5200 may comprise a first through-hole or aperture 5201 and a secondthrough-hole or aperture 5202. Each of the two through-holes 5201, 5202may be connected to a fluidic channel on a bottom layer of the fluidicdevice. The first and second through-holes 5201, 5202 may penetrate thechip surface and be in communication with a fluidic channel locatedbelow the reservoir 5200 as described herein. The fluidic channel mayfor example be an elution channel as described herein. The firstthrough-hole 5201 and the second through-hole 5202 may open ontodifferent plane within the reservoir 5200. The first through-hole 5201may be an elliptical through-hole 5201 dimensioned such that a pipettetip can be easily positioned for reliable fluid recovery from thechannel of the device. The first and second through-holes 5201, 5202 maybe generally aligned with each other and with a longitudinal axis of thefluidic channel such that application of a fluid into the channel viathe second through-hole 5202 pushes the fluid along the channel towardsthe first through-hole 5201 as described herein. The second through-hole5202 may be used to create a fluidic connection and electricalconnection to a high-voltage electrode. As shown in FIG. 52B, a pipettetip 5203 can be inserted into the first through-hole 5201 untilinterference between pipette tip and one or more walls of the firstthrough-hole 5201 prevents further insertion at a defined couplingposition. The coupling position can be defined to ensure an unrestrictedpath between the fluid volume of the fluidic device and the inlet holeof the pipette tip. Retraction force may be reduced by minimizing pointsof contact to two points 5204. Shown in FIG. 52C, the major and minoraxes of the first through-hole 5201 can be dimensioned such that, whenthe coupling position is achieved, a fluidic pathway 5205 is maintainedaround the pipette tip 5203.

The first through-hole 5201 may for example have an elliptical shape.Alternatively, the first through-hole 5201 may be circular. The firstthrough-hole 5201 may have any shape desired by one or ordinary skill inthe art.

The second through-hole 5202 may for example have an elliptical shapeAlternatively, the second through-hole 5202 may be circular. The secondthrough-hole 5202 may have any shape desired by one or ordinary skill inthe art.

The first through-hole 5201 may have a diameter within a range of about100 um to about 3 mm.

The first through-hole 5201 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

The second through-hole 5202 may have a diameter within a range of about100 um to about 3 mm.

The second through-hole 5202 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

The first through-hole 5201 may be an elongate through-hole thatpenetrates the surface of the chip to couple the reservoir 5200 to thechannel. The first through-hole 5201 may, for example, be cylindrical asdescribed herein. The first through-hole 5201 may, for example, have aheight from the channel to the reservoir 5200 within a range of about 1mm to about 3 mm.

The second through-hole 5202 may be an elongate through-hole thatpenetrates the surface of the chip to couple the reservoir 5200 to thechannel. The second through-hole 5202 may, for example, be cylindricalas described herein.

The second through-hole 5202 may have a height above the channel greaterthan the height above the channel of the first through-hole 5201.

The reservoir 5200 may comprise a circular cross-section. Alternatively,the reservoir 5200 may comprise an elongate or oval cross-section.

In some embodiments, the channel below the reservoir 5200 may comprise abreak as shown in FIG. 50A1 and the first and second through-holes 5201,5202 may be in fluidic and/or electrical communication with first andsecond terminating ends of the channel as described herein. The firstand second through-holes 5201, 5202 may be positioned at differentheights within the reservoir. The first through-hole 5201 may enter thereservoir 5200 at a position lower in the reservoir than the secondthrough-hole 5102. The first and second through-holes 5201, 5202 may beseparated by a step or platform 5206 as described herein.

The reservoir 5200 may be circular. The reservoir 5200 may have adiameter of about 5.3 mm.

The reservoir 5200 may be defined by a wall having a height of no morethan 25 mm, no more than 15 mm, no more than 10 mm, or greater than 10mm. The reservoir 5200 may have height of about 6 mm. The reservoir 5200may have height within a range of about 8 mm to about 10 mm.

The first and second through holes 5201, 5202 may have areas within arange of about 0.2 mm² to 7 mm2. The first through-hole 5201 may have anarea within a range of about 1 mm² to about 2.75 mm². For example thesecond through-hole 5202 may have an area within a range of about 0.8mm² to about 1.5 mm².

The through-holes 5201, 5202 may have substantially the same shape. Thethrough-holes 5201, 5202 may have different shapes.

The first through-hole 5201 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel or the width of the reservoir 5200.

The second through-hole 5202 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel or the width of the reservoir 5200.

The through-holes 5201, 5202 may be arranged along a longitudinal axisof the channel 5003.

The first through-hole 5201 may have a maximum distance across (i.e.length) of about 1.75 mm and a minimum distance across (i.e. width) ofabout 1.3 mm.

The second through-hole 5202 may be relatively circular with a diameterof about 1.2 mm. The second through-hole 5202 may be shaped so as tospread the electrical field evenly across the reservoir 5200.

The second through-hole 5202 may enter the reservoir though a platform5126 in the reservoir 5200 positioned about 1 mm to about 6 mm above apoint of entry into the reservoir 5200 of the first through-hole 5201.

The volume of the reservoir 5200 between the first and secondthrough-holes 5201, 5202 may be no more than about 2.5 ml, 1 ml, or 0.5ml. The volume of the reservoir 5200 between the first and secondthrough-holes 5201, 5202 may be about 0.1 mL.

At least part of each through-hole 5201, 5202 may be substantiallyco-extensive with the fluidic channel across said width of said fluidicchannel.

The first through-hole 5201 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel and/or awidth of the reservoir 5200. The at least one side(s) may (each) be atleast 75% linear.

The second through-hole 5202 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel and/or awidth of the reservoir 5200. The at least one side(s) may (each) be atleast 75% linear.

The first and second through-holes 5201, 5202 may have a shape that,when said fluidic channel and said reservoir 5200 comprise anelectrically conductive fluid and an electric current is passed throughsaid fluidic channel, at least 5%, at least 6%, at least 7%, at least10%, or at least 20% of said electric current passes through saidreservoir 5200 between the first and the second through-holes 5201,5202.

FIG. 52D shows a comparison between a reservoir 5200 with a circularfirst through-hole 5201 pipette interface (n=2) and one with anelliptical first through-hole 5201 interface (n=2) for fluidic volumerecovery. The circular through-hole design served as a control wherebythe pipette tip was not constrained to a precise coupling position. Thepresent disclosure provides, in some embodiments, a fluidic reservoir5200 with an elliptical through-hole interface 5201 that may allow forthe precise positioning of a pipette for maximized fluid evacuation. Thefluidic reservoir 5200 may be incorporated in a larger fluidic device.Positioning may be achieved by the constraining of the pipette tip uponinsertion to a final coupling position. The elliptical geometry of thethrough-hole 5201 may allow unrestricted fluid flow around the pipettetip and may reduce the force required to retract the pipette tip fromits coupling position. Retraction force may be minimized by the designlimiting contact between pipette tip and reservoir through-hole 5201 totwo points 5204. Minimized retraction force mitigates the risk ofdisturbing the position of the fluidic device upon retraction.

FIG. 53A shows an exemplary fluidic reservoir 5300. The reservoir 5300may be substantially similar to reservoir 5200 but with rounded edge5302 that may favor drainage toward the first through-hole extractionsite 5301. FIG. 53B depicts an alternate view of reservoir 5300. FIG.53C shows a comparison between a reservoir with angular interior edges(e.g. reservoir 5200) and one with filleted interior edges 5304 (e.g.reservoir 5300) for fluidic volume recovery.

FIG. 53D shows a top view of an exemplary elution reservoir 5310. FIG.53E shows a section view of the elution reservoir 5310. The reservoir5310 may be located on any of the chip devices described herein. Thereservoir 5310 may have an elongate or oval cross-section. The reservoir5310 may be configured to hold a volume of elution buffer as describedherein. Upon completion of an ITP run, the target nucleic acids mayreside within this volume as described herein. The reservoir 5310 maycomprise a first through-hole or aperture 5311 and a second through-holeor aperture 5312. Each of the two through-holes 5311, 5312 may beconnected to a fluidic channel 5313 on a bottom layer of the fluidicdevice. The first and second through-holes 5311, 5312 may penetrate thechip surface and be in communication with a fluidic channel 5313 locatedbelow the reservoir 5310 as described herein. The fluidic channel 5313may for example be an elution channel as described herein. The firstthrough-hole 5311 and the second through-hole 5311 may be on the sameplane within the reservoir 5310. The first through-hole 5311 may bedimensioned such that a pipette tip can be easily positioned forreliable fluid recovery from said device. For example, the firstthrough-hole 5311 may comprise a guide wall 5314 configured to guideplacement of the pipette tip toward the channel 5313. The guide wall5314 may be configured to constrain the pipette tip orientation andposition to properly align the pipette tip with the channel 5313. Theguide wall 5314 may, for example, have a narrower region 5315 located atthe channel 5313 and the wider region of 5316 located at the entry wayfor ambient air of the loading reservoir 5300. The wider portion 5316and the narrower portion 5315 may have a D-shaped cross-section. Theguide wall 5314 may taper between the wider portion 5316 and thenarrower portion 5315 at an angle configured to guide the pipette tochannel 5313.

The first and second through-holes 5311, 5312 may be generally alignedwith each other and with a longitudinal axis of the fluidic channel 5313such that application of a fluid into the channel 5313 via the secondthrough-hole 5312 pushes the fluid along the channel 5313 towards thefirst through-hole 5311 as described herein. The second through-hole5312 may be used to create a fluidic connection and electricalconnection to a high-voltage electrode.

The first through-hole 5311 may for example have a D-shapedcross-section. Alternatively, the first through-hole 5311 may have anelliptical shape. Alternatively, the first through-hole 5311 may becircular. The first through-hole 5311 may have any shape desired by oneor ordinary skill in the art.

The second through-hole 5312 may for example have a D-shapedcross-section. Alternatively, the second through-hole 5312 may have anelliptical shape Alternatively, the second through-hole 5312 may becircular. The second through-hole 5312 may have any shape desired by oneor ordinary skill in the art.

The first through-hole 5211 may have a diameter within a range of about100 um to about 3 mm.

The wider portion 5316 of the first through-hole 5211 may have adiameter within a range of about 100 um to about 3 mm.

The narrower portion 5315 of the first through-hole 5211 may have adiameter within a range of about 100 um to about 3 mm.

The first through-hole 5311 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

The guide wall 5314 may taper between the wider portion 5316 and thenarrower portion 5315 at an angle configured to guide the pipette tochannel 5313. The guide wall 5314 may taper at an angle within a rangeof about 60 degrees to about 90 degrees.

The second through-hole 5312 may have a diameter within a range of about100 um to about 3 mm.

The second through-hole 5312 may have a maximum dimension across of lessthan about 1.5 mm, for example less than about 1 mm.

The first through-hole 5311 may be an elongate through-hole thatpenetrates the surface of the chip to couple the reservoir 5310 to thechannel. The first through-hole 5311 may, for example, be tapered asdescribed herein. The first through-hole 5311 may, for example, have aheight from the channel to the reservoir 5310 within a range of about 1mm to about 3 mm.

The second through-hole 5312 may be an elongate through-hole thatpenetrates the surface of the chip to couple the reservoir 5310 to thechannel. The second through-hole 5312 may, for example, be cylindricalas described herein.

The reservoir 5310 may comprise a circular cross-section. Alternatively,the reservoir 5310 may comprise an elongate or oval cross-section.

In some embodiments, the channel 5313 below the reservoir 5310 maycomprise a break as shown in FIG. 50A1 and the first and secondthrough-holes 5311, 5312 may be in fluidic and/or electricalcommunication with first and second terminating ends of the channel 5313as described herein. The first and second through-holes 5311, 5312 maybe positioned at different heights within the reservoir 5310. The firstthrough-hole 5311 may enter the reservoir 5310 at a position lower inthe reservoir than the second through-hole 5312. The first and secondthrough-holes 5311, 5312 may be separated by a step or platform asdescribed herein.

The reservoir 5310 may comprise an elongate shape, for example anelliptical shape, having a maximum diameter (i.e. length) across ofabout 7 mm and a minimum diameter across (i.e. width) of about 3.4 mm.

The reservoir 5310 may be defined by a wall having a height of no morethan 25 mm, no more than 15 mm, no more than 10 mm, or greater than 10mm. The reservoir 5310 may have height of about 6 mm. The reservoir 5310may have height within a range of about 8 mm to about 10 mm.

The first and second through holes 5311, 5312 may have areas within arange of about 0.2 mm² to 7 mm2. The first through-hole 5311 may have anarea within a range of about 1 mm² to about 2.75 mm². For example thesecond through-hole 5312 may have an area within a range of about 0.8mm² to about 1.5 mm².

The through-holes 5311, 5312 may have substantially the same shape. Thethrough-holes 5311, 5312 may have different shapes.

The first through-hole 5311 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel 5313 or the width of the reservoir 5310. The firstthrough-hole 5311 may be D-shaped.

The second through-hole 5312 may have a shape of a square, a rectangle,an oval, or have an elongated direction in a direction of the width ofthe channel 5313 or the width of the reservoir 5310.

The through-holes 5311, 5312 may be arranged along a longitudinal axisof the channel 5313.

The first through-hole 5311 may have a maximum distance across (i.e.length) of about 1.75 mm and a minimum distance across (i.e. width) ofabout 1.3 mm.

The second through-hole 5312 may be relatively circular with a diameterof about 1.2 mm. The second through-hole 5312 may be shaped so as tospread the electrical field evenly across the reservoir 5310.

The volume of the reservoir 5310 between the first and secondthrough-holes 5311, 5312 may be no more than about 2.5 ml, 1 ml, or 0.5ml. The volume of the reservoir 5200 between the first and secondthrough-holes 5311, 5312 may be about 0.1 mL.

At least part of each through-hole 5311, 5312 may be substantiallyco-extensive with the fluidic channel 5313 across said width of saidfluidic channel 5313.

The first through-hole 5311 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel 5313 and/ora width of the reservoir 5310. The at least one side(s) may (each) be atleast 75% linear.

The second through-hole 5312 may comprise at least one side, for exampleone or two sides, that span the width of the fluidic channel 5313 and/ora width of the reservoir 5310. The at least one side(s) may (each) be atleast 75% linear.

The first and second through-holes 5311, 5312 may have a shape that,when said fluidic channel and said reservoir 5310 comprise anelectrically conductive fluid and an electric current is passed throughsaid fluidic channel, at least 5%, at least 6%, at least 7%, at least10%, or at least 20% of said electric current passes through saidreservoir 5310 between the first and the second through-holes 5311,5312.

In some embodiments, this disclosure provides a fluidic reservoir withrounded interior edges that maximize drainage toward a through-hole uponextraction of fluid from the through-hole. The rounded edges maximizerecovery of fluid from the reservoir by reducing the fluid volumerequired to wet between the horizontal floor and vertical walls of thereservoir. Additionally, the rounded edges minimize wetted surface areainside of the reservoir, improving recovery of fluid.

Channels on a fluidic device can be closed. For example, a mechanicalactuator coupled to a mechanical member can be used to apply pressure tocompletely or partially close a channel (e.g., by deformation of thechannel). Elution reservoirs can be closed off from the ITP channel todefine a fixed elution volume. Channel closing can result in reducedflow or completely blocked flow. Channel closing can result in increasedresistance to fluid flow. In some instances, channel closing canincrease fluidic resistance by a factor of at least 2, 3, 4, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, or 100.

FIG. 12A shows an exemplary mechanical member 1200 which can be used toapply pressure to close or at least partially close the channels 1201 ofa fluidic device 1210, the fluidic device 1210 comprising multiplechannels in parallel (for example the device of FIG. 6C comprising eightindependent parallel channels). The mechanical member 1200 can comprisea comb-like structure with teeth 1202 that line up with two locations1204, 1205 in each of the eight channels of the chip as shown in FIG.12B. Mechanical pressure can be applied by the teeth 1202 to permanentlyor plastically close or at least partially close the channels 1201 tolimit, reduce, or prevent liquid flow to or from the elution reservoirs1203 and control the elution volume. At least partially closing thechannels 1201 may increase resistance to fluid flow between the channels1201 and the elution reservoirs 1203. The mechanical member 1200 may becoupled to a mechanical actuator which generates the force applied tothe channel 1201 by the teeth 1202 of the mechanical member 1200. Themechanical member 1200 may comprise a material with a Young's modulus ofelasticity greater than a Young's modulus of elasticity of the channel1201. One or more teeth 1202 of the mechanical member 1200 may beconfigured to heat a channel 1201. One or more teeth 1202 of themechanical member 1200 may be thermally coupled to a heater or heatingelement. The mechanical member 1200 may optionally comprise a heater orheating element. Heat can optionally be applied by the teeth 1202 topermanently or plastically close the channels 1201. One or more teeth1202 may be heated to a temperature greater than the glass transitiontemperature of at least one wall of one or more channels 1201. FIG. 12Cshows how the sixteen teeth 1202 of the mechanical member 1200 line upwith the sixteen locations 1204, 1205 on the chip 1210 (two perchannel). Each tooth 1202 may be configured to deliver mechanicalpressure to the channel 1201 in order to plastically deform at least onewall of the channel 1201. Each channel 1201 is contacted by themechanical member 1200 and plastically deformed at a first closelocation 1204 and a second close location 1205 to isolate the elutionreservoir volume and increase fluid resistance between the channel 1201and the reservoir 1203. In some instances, a tooth 1202 may applymechanical pressure to the channel location 1204 upstream of thereservoir 1203. In some instances, a tooth 1202 may apply mechanicalpressure to a junction where the reservoir 1203 and the channel 1201meet. In some instances, a tooth 1202 may apply mechanical pressure to ajunction 1205 where the reservoir and a buffering channel meet toprevent fluid communication between the reservoir 1203 and a bufferingreservoir.

In some cases, the mechanical member 1200 may comprise one tooth 1202per channel which aligns with the first close location 1204. Forexample, the channel shown in FIG. 5A does not comprise a buffer channelor reservoir connected to the elution reservoir and thus may not need asecond close location 1205 beyond the elution reservoir. In some cases,the mechanical member 1200 is configured to close each of the channels1201 on a chip 1210 at one or more locations. In some cases, themechanical member 1200 is configured to leave one or more channel 1201on the chip 1210 open such that only a fraction of channels 1201 on thechip 1210 are closed.

The mechanical member 1200 may apply a force of at least 0.25 lbs perchannel via teeth 1202. Each tooth 1202 of the mechanical member 1200may apply a force of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, or 5pounds to a channel 1201.

FIG. 54A shows an exemplary mechanical member 1220 which can be used toapply pressure to close or at least partially close the channels 1201 ofa fluidic device 1210, the fluidic device 1210 comprising multiplechannels in parallel (for example the device of FIG. 6C comprising eightindependent parallel channels). The mechanical member 1220 can comprisea ridge-like structure similar to that of mechanical member 1200 butwithout teeth. The ridge-like structure may extend across and compresseach of the eight channels of the chip at two locations 1204, 1205 asshown in FIG. 54B. Mechanical pressure can be applied by the ridge 1222to permanently or plastically close or at least partially close thechannels 1201 in order to limit, reduce, or prevent liquid flow to orfrom the elution reservoirs 1203 and control the elution volume. Atleast partially closing the channels 1201 may increase resistance tofluid flow between the channels 1201 and the elution reservoirs 1203.The mechanical member 1220 may be coupled to a mechanical actuator 1213which generates the force applied to the channel 1201 by the ridge 1222of the mechanical member 1220. The mechanical member 1220 may comprise amaterial with a Young's modulus of elasticity greater than a Young'smodulus of elasticity of the channel 1201. The ridge-like structure 1222of the mechanical member 1220 may be configured to heat a channel 1201.The ridge-like structure 1222 of the mechanical member 1220 may bethermally coupled to a heater or heating element. The mechanical member1220 may optionally comprise a heater or heating element. Heat canoptionally be applied by the ridge-like structure 1222 to permanently orplastically close the channels 1201. The ridge-like structure 1222 maybe heated to a temperature greater than the glass transition temperatureof at least one wall of one or more channels 1201. FIG. 54C shows howthe ridge 1222 of the mechanical member 1220 line up with the sixteenlocations 1204, 1205 on the chip 1210 (two per fluidic channel). Theridge 1222 may be configured to deliver mechanical pressure to thechannel 1201 in order to plastically deform at least one wall of thechannel 1201. Each channel 1201 is contacted by the mechanical member1220 and plastically deformed at a first close location 1204 and asecond close location 1205 to isolate the elution reservoir volume andincrease fluid resistance between the channel 1201 and the reservoir1203. In some instances, the ridge structure 1222 may apply mechanicalpressure to the channel location 1204 upstream of the reservoir 1203. Insome instances, the ridge structure 1222 may apply mechanical pressureto a junction where the reservoir 1203 and the channel 1201 meet. Insome instances, the ridge structure 1222 may apply mechanical pressureto a junction 1205 where the reservoir and a buffering channel meet toprevent fluid communication between the reservoir 1203 and a bufferingreservoir.

FIG. 54D shows a break out component assembly diagram for the mechanicalactuator coupled to a ridge-like structure 1220 for closing channels.This assembly may be a stand-alone assembly or incorporated as asub-assembly of any of the instruments described herein. This design maybe configured to be modular and can accommodate different memberstructure designs including the tooth or ridge, or alternate structuresas will be apparent to one of ordinary skill in the art.

In some embodiments, any of the mechanical members described herein maybe triggered to close one or more channels by the cessation of theelectrical field applied to one or more channels of the device.

FIG. 55 depicts a pneumatic control block diagram for beta prototype andproduction instrument.

FIGS. 56A-56B and 58A-58C show alternative mechanisms for closingchannels without the use of heat or pressure-induced plastic deformationof a fluidic device. A physical member may be used to close or partiallyclose the channels of the fluidic device by applying a membrane thatprovides a seal to chip reservoirs on a fluidic device. The mechanismsmay use adhesion or self-healing of membrane, with or without appliedmechanical pressure, to close or partially close the channels withoutcausing deformation of the chip device or material. The mechanism mayapply constant force and/or pressure passively (e.g. by applying a fixedmechanical load) to the fluid reservoirs of a fluidic device.

FIGS. 56A-56B show an exemplary mechanical member 5600 which can be usedto close or at least partially close the reservoirs 5604 of a fluidicdevice 5605, the fluidic device 5605 comprising multiple channels inparallel (e.g. 8 parallel channels in a fluidic device). The mechanicalmember 5600 can comprise a compliant structure 5602 that lines up with aseries of reservoirs connected to an individual channel in a fluidicdevice (e.g. 5 reservoirs). The elution reservoir 5603 is intentionallyleft open to the atmosphere to allow extraction (e.g. by pipetting) ofmaterial processed in the fluidic device 5605 while the mechanicalmember 5600 is closing or partially closing the other five reservoirs5604. Mechanical pressure can be applied to the compliant structure 1502to temporarily close or at least partially close the reservoirs 5604 tolimit, reduce, or prevent liquid flow to or from the elution reservoirs5603 and control the elution volume. At least partially closing thereservoirs 5604 may increase resistance to fluid flow between thesample/buffer reservoirs 5604 and the elution reservoirs 5603.

The compliant structure 5602 may comprise or be similar to a PCR filmseal or membrane. A PCR film seal is a type of disposable membrane thatcan be adhesively applied over the fluidic reservoirs to seal them andprevent flow in the connected channels. The process of adhesion may beimproved with applied mechanical pressure. Mechanical pressure may beapplied so as to not apply pressure to the fluids in the reservoir inorder to prevent or minimize liquid flowing within the chip duringchannel closing. A PCR film/membrane may represent a low-cost,disposable option for channel closing that may reducecross-contamination which can sometimes occur with repeated applicationof the same membrane (i.e. a reusable membrane) from chip to chip.

The compliant structure 5602 may comprise or be similar to a compliantor self-healing membrane seal. A compliant or self-healing membrane sealis a type of disposable or reusable membrane that can be applied with orwithout mechanical pressure over the fluidic reservoirs to seal them andprevent or reduce flow in the connected channels. One type of such sealis a compliant rubber gasket material. This rubber sealing member can bedisposable or re-usable. A reusable rubber sealing member may requirecleaning of the membrane material between uses with a compatiblecleaning solvent in order to prevent or reduce the risk ofcross-contamination between uses. The compliant rubber seal may beapplied with a fixed mechanical load or pressure. The mechanicalpressure may be applied so as to not apply pressure to the fluids in thereservoir in order to prevent or minimize liquid flowing within the chipduring channel closing.

FIG. 57 shows an exemplary mechanical member 5700 which can be used toapply pressure to close or at least partially close the reservoirs 5704of a fluidic device 5705, the fluidic device 5705 comprising multiplechannels in parallel. The mechanical member 5700 can comprise a loadbearing structure 5701 and compliant structure 5702 that lines up withopen buffer or sample reservoirs on the fluidic device. The elutionreservoir 5703 on the fluidic device is intentionally left open toatmosphere to allow extraction of material processed in the fluidicdevice 5705 while the mechanical member is closing or partially closingthe other five reservoirs 5704. Mechanical pressure can be applied bythe rigid structure 5701 to the compliant structure 5702 to temporarilyclose or at least partially close the reservoirs 5704 to limit, reduce,or prevent liquid flow to or from the elution reservoirs 5703 andcontrol the elution volume. At least partially closing the reservoirs5704 may increase resistance to fluid flow between the sample/bufferreservoirs 5704 and the elution reservoirs 5703.

FIGS. 58A-58C show an exemplary channel closer with rubber sealingmember and mechanical actuator to provide a mechanical load for sealing.FIG. 58A shows an image of the side view of a channel closing device5800 comprising a compliant rubber sealing member 5801 and a structurefor applying mechanical load 5802 to close or at least-partially closethe reservoirs 5803 of a fluidic device. Varying materials (e.g.polyurethane, silicone, vinyl), durometers (Extra soft to soft, or30OO-50 durometer), and thicknesses (e.g. 0.05″ to 0.25″) may beemployed as will be understood by one of ordinary skill in the art. Inone example, the rubber may be polyurethane with a durometer of 40OO anda thickness of 0.060″. The rubber member 5801 can be configured to coverall reservoirs 5803 but the elution reservoir(s) 5805 on the fluidicdevice. The rubber member 5801 can be mounted to a flat, solid support5804 (e.g. plastic, glass, or a metal such as aluminum) in order toprovide a flat sealing surface to the chip reservoirs 5801. A mechanicalload 5802 (such as about 0 to about 2 kg) can be applied to the flatsupport 5804 by a mechanical actuator in order to further compress thecompliant rubber member 5801 and provide sealing to the chip reservoirs5803. Alternatively or in combination, the solid support element 5804,for example a weighted solid support, may be used to passively apply themechanical load to the rubber member 5801.

FIGS. 58B and 58C show two exemplary configurations for the rubbermember 5801. FIG. 58B shows an example wherein a rubber membrane 5801 isconfigured to specifically interface only with the tops of thereservoirs 5803 on the top of the fluidic device (as opposed tointerfacing with every structure at the top of the device). FIG. 58Cshows an example wherein the rubber membrane 5801 is configured as asimple sheet or layer and generally interfaces with all structures onthe top of the device but elution reservoir(s) 5805.

Channels on a fluidic device (e.g., sample preparation zones,isotachophoresis zones) can have a large enough width, height, ordiameter such that contaminants, such as embedding material (e.g.,paraffin), can deposit on the channel walls while still leaving adequateroom for fluid flow within the channel. In some cases, a channel on afluidic device has a width, height, or diameter of less than or equal to20 millimeters (mm), 19 mm, 18 mm, 17 mm, 16 mm, 15 mm, 14 mm, 13 mm, 12mm, 11 mm, 10 mm, 9 mm, 8 mm 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm,0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1mm. In some cases, a channel on a fluidic device has a width, height, ordiameter of at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19mm, or 20 mm. In some cases, a channel on a fluidic device has a widthwithin a range of about 1 mm to about 3.8 mm. In some cases, a channelon a fluidic device has a height within a range of about 0.1 mm to about1.2 mm.

Sample channels on the fluidic device can have a height within a rangeof about 10 um to about 2 mm, for example within a range of about 400 umto about 1.2 mm. In some cases, one or more sample channels on thefluidic device can have a height within a range bounded by any two ofthe following values: 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um,80 um, 90 um, 100 um, 110 um, 120 um, 130 um, 140 um, 150 um, 160 um,170 um, 180 um, 190 um, 200 um, 200 um, 210 um, 220 um, 230 um, 240 um,250 um, 260 um, 270 um, 280 um, 290 um, 300 um, 310 um, 320 um, 330 um,340 um, 350 um, 360 um, 370 um, 380 um, 390 um, 400 um, 410 um, 420 um,430 um, 440 um, 450 um, 460 um, 470 um, 480 um, 490 um, 500 um, 510 um,520 um, 530 um, 540 um, 550 um, 560 um, 570 um, 580 um, 590 um, 600 um,610 um, 620 um, 630 um, 640 um, 650 um, 660 um, 670 um, 680 um, 690 um,700 um, 710 um, 720 um, 730 um, 740 um, 750 um, 760 um, 770 um, 780 um,790 um, 800 um, 810 um, 820 um, 830 um, 840 um, 850 um, 860 um, 870 um,880 um, 890 um, 900 um, 910 um, 920 um, 930 um, 940 um, 950 um, 960 um,970 um, 980 um, 990 um, 1000 um, 1010 um, 1020 um, 1030 um, 1040 um,1050 um, 1060 um, 1070 um, 1080 um, 1090 um, 1100 um, 1110 um, 1120 um,1130 um, 1140 um, 1150 um, 1160 um, 1170 um, 1180 um, 1190 um, 1200 um,1210 um, 1220 um, 1230 um, 1240 um, 1250 um, 1260 um, 1270 um, 1280 um,1290 um, 1300 um, 1310 um, 1320 um, 1330 um, 1340 um, 1350 um, 1360 um,1370 um, 1380 um, 1390 um, 1400 um, 1410 um, 1420 um, 1430 um, 1440 um,1450 um, 1460 um, 1470 um, 1480 um, 1490 um, 1500 um, 1510 um, 1520 um,1530 um, 1540 um, 1550 um, 1560 um, 1570 um, 1580 um, 1590 um, 1600 um,1610 um, 1620 um, 1630 um, 1640 um, 1650 um, 1660 um, 1670 um, 1680 um,1690 um, 1700 um, 1710 um, 1720 um, 1730 um, 1740 um, 1750 um, 1760 um,1770 um, 1780 um, 1790 um, 1800 um, 1810 um, 1280 um, 1830 um, 1840 um,1850 um, 1860 um, 1870 um, 1880 um, 1890 um, 1900 um, 9110 um, 1920 um,1930 um, 1940 um, 1950 um, 1960 um, 1970 um, 1980 um, 1990 um, and 2000um

Leading electrolyte buffer channels on the fluidic device can have aheight within a range of about 10 um to about 1 mm, for example lessthan about 600 um. In some cases, one or more leading electrolyte bufferchannels on the fluidic device can have a height within a range boundedby any two of the following values: 10 um, 20 um, 30 um, 40 um, 50 um,60 um, 70 um, 80 um, 90 um, 100 um, 110 um, 120 um, 130 um, 140 um, 150um, 160 um, 170 um, 180 um, 190 um, 200 um, 200 um, 210 um, 220 um, 230um, 240 um, 250 um, 260 um, 270 um, 280 um, 290 um, 300 um, 310 um, 320um, 330 um, 340 um, 350 um, 360 um, 370 um, 380 um, 390 um, 400 um, 410um, 420 um, 430 um, 440 um, 450 um, 460 um, 470 um, 480 um, 490 um, 500um, 510 um, 520 um, 530 um, 540 um, 550 um, 560 um, 570 um, 580 um, 590um, 600 um, 610 um, 620 um, 630 um, 640 um, 650 um, 660 um, 670 um, 680um, 690 um, 700 um, 710 um, 720 um, 730 um, 740 um, 750 um, 760 um, 770um, 780 um, 790 um, 800 um, 810 um, 820 um, 830 um, 840 um, 850 um, 860um, 870 um, 880 um, 890 um, 900 um, 910 um, 920 um, 930 um, 940 um, 950um, 960 um, 970 um, 980 um, 990 um, and 1000 um.

Elution channels on the fluidic device can have a height within a rangeof about 10 um to about 1 mm, for example less than about 600 um. Insome cases, one or more elution buffer channels on the fluidic devicecan have a height within a range bounded by any two of the followingvalues: 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um,100 um, 110 um, 120 um, 130 um, 140 um, 150 um, 160 um, 170 um, 180 um,190 um, 200 um, 200 um, 210 um, 220 um, 230 um, 240 um, 250 um, 260 um,270 um, 280 um, 290 um, 300 um, 310 um, 320 um, 330 um, 340 um, 350 um,360 um, 370 um, 380 um, 390 um, 400 um, 410 um, 420 um, 430 um, 440 um,450 um, 460 um, 470 um, 480 um, 490 um, 500 um, 510 um, 520 um, 530 um,540 um, 550 um, 560 um, 570 um, 580 um, 590 um, 600 um, 610 um, 620 um,630 um, 640 um, 650 um, 660 um, 670 um, 680 um, 690 um, 700 um, 710 um,720 um, 730 um, 740 um, 750 um, 760 um, 770 um, 780 um, 790 um, 800 um,810 um, 820 um, 830 um, 840 um, 850 um, 860 um, 870 um, 880 um, 890 um,900 um, 910 um, 920 um, 930 um, 940 um, 950 um, 960 um, 970 um, 980 um,990 um, and 1000 um.

In some cases, a channel on a fluidic device has a length of at leastabout 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 25mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190mm, 200 mm, 210 mm, 220 mm, 230 mm, 240 mm, 250 mm, 260 mm, 270 mm, 280mm, 290 mm, 300 mm, 310 mm, 320 mm, 330 mm, 340 mm, 350 mm, 360 mm, 370mm, 380 mm, 390 mm, 400 mm, 410 mm, 420 mm, 430 mm, 440 mm, 450 mm, 460mm, 470 mm, 480 mm, 490 mm, or 500 mm. In some cases, a channel on afluidic device has a length of less than or equal to about 500 mm, 490mm, 480 mm, 470 mm, 460 mm, 450 mm, 440 mm, 430 mm, 420 mm, 410 mm, 400mm, 390 mm, 380 mm, 370 mm, 360 mm, 350 mm, 340 mm, 330 mm, 320 mm, 310mm, 300 mm, 290 mm, 280 mm, 270 mm, 260 mm, 250 mm, 240 mm, 230 mm, 220mm, 210 mm, 200 mm, 190 mm, 180 mm, 170 mm, 160 mm, 150 mm, 140 mm, 130mm, 120 mm, 110 mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 45 mm, 40mm, 35 mm, 30 mm, 25 mm, 20 mm, 19 mm, 18 mm, 17 mm, 16 mm, 15 mm, 14mm, 13 mm, 12 mm, 11 mm, 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3mm, 2 mm, or 1 mm.

Channels on a fluidic device can have a large enough width, height, ordiameter so as to accommodate a large sample volume. In some cases, achannel on a fluidic device has a width greater than its height so as toreduce a temperature rise due to Joule heating in the channel. In somecases, a channel on a fluidic device has a ratio of width to height ofat least 2:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1,55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1. In somecases, a channel on a fluidic device has a ratio of width to height ofat most 2:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1,55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1. In somecases, a channel on a fluidic device has a cross-sectional area lessthan about 0.1 mm², 0.2 mm², 0.3 mm², 0.4 mm², 0.5 mm², 0.6 mm², 0.7mm², 0.8 mm², 0.9 mm², 1 mm², 1.1 mm², 1.2 mm², 1.3 mm², 1.4 mm², 1.5mm², 1.6 mm², 1.7 mm², 1.8 mm², 1.9 mm², 2 mm², 2.1 mm², 2.2 mm², 2.3mm², 2.4 mm², 2.5 mm², 2.6 mm², 2.7 mm², 2.8 mm², 2.9 mm², 3 mm², 3.1mm², 3.2 mm², 3.3 mm², 3.4 mm², 3.5 mm², 3.6 mm², 3.7 mm², 3.8 mm², 3.9mm², 4 mm², 4.1 mm², 4.2 mm², 4.3 mm², 4.4 mm², 4.5 mm², 4.6 mm², 4.7mm², 4.8 mm², 4.9 mm², 5 mm², 6 mm², 7 mm², 8 mm², 9 mm², 10 mm², 11mm², 12 mm², 13 mm², 14 mm², or 15 mm². In some cases, a channel on afluidic device has a cross-sectional area more than about 0.1 mm², 0.2mm², 0.3 mm², 0.4 mm², 0.5 mm², 0.6 mm², 0.7 mm², 0.8 mm², 0.9 mm², 1mm², 1.1 mm², 1.2 mm², 1.3 mm², 1.4 mm², 1.5 mm², 1.6 mm², 1.7 mm², 1.8mm², 1.9 mm², 2 mm², 2.1 mm², 2.2 mm², 2.3 mm², 2.4 mm², 2.5 mm², 2.6mm², 2.7 mm², 2.8 mm², 2.9 mm², 3 mm², 3.1 mm², 3.2 mm², 3.3 mm², 3.4mm², 3.5 mm², 3.6 mm², 3.7 mm², 3.8 mm², 3.9 mm², 4 mm², 4.1 mm², 4.2mm², 4.3 mm², 4.4 mm², 4.5 mm², 4.6 mm², 4.7 mm², 4.8 mm², 4.9 mm², 5mm², 6 mm², 7 mm², 8 mm², 9 mm², 10 mm², 11 mm², 12 mm², 13 mm², 14 mm²,or 15 mm². In some cases, a channel on a fluidic device has a minimumlength scale for heat dissipation less than about 1 micrometer (μm), 5μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm,300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, or 600 μm. In somecases, a channel on a fluidic device has a minimum length scale for heatdissipation more than about 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30μm, 40 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400μm, 450 μm, 500 μm, 550 μm, or 600 μm.

In some cases, a channel on a fluid device has a total volume of atleast about 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL,70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 175 μL, 200 μL, 225 μL, 250 μL, 275μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL,10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20mL, 25 mL, 30 mL, 35 mL, 40 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75mL, 80 mL, 85 mL, 90 mL, 95 mL, or 100 mL. In some cases, a channel on afluid device has a total volume of at most about 1 microliter (μL), 10μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150μL, 175 μL, 200 μL, 225 μL, 250 μL, 275 μL, 300 μL, 350 μL, 400 μL, 450μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95mL, or 100 mL.

Sample channels on the fluidic device can have a volume within a rangeof about 10 uL to about 1 mL, for example within a range of about 50 uLto about 500 uL. In some cases, one or more sample channel on thefluidic device can have a volume within a range bounded by any two ofthe following values: 10 uL, 20 uL, 30 uL, 40 uL, 50 uL, 60 uL, 70 uL,80 uL, 90 uL, 100 uL, 110 uL, 120 uL, 130 uL, 140 uL, 150 uL, 160 uL,170 uL, 180 uL, 190 uL, 200 uL, 200 uL, 210 uL, 220 uL, 230 uL, 240 uL,250 uL, 260 uL, 270 uL, 280 uL, 290 uL, 300 uL, 310 uL, 320 uL, 330 uL,340 uL, 350 uL, 360 uL, 370 uL, 380 uL, 390 uL, 400 uL, 410 uL, 420 uL,430 uL, 440 uL, 450 uL, 460 uL, 470 uL, 480 uL, 490 uL, 500 uL, 510 uL,520 uL, 530 uL, 540 uL, 550 uL, 560 uL, 570 uL, 580 uL, 590 uL, 600 uL,610 uL, 620 uL, 630 uL, 640 uL, 650 uL, 660 uL, 670 uL, 680 uL, 690 uL,700 uL, 710 uL, 720 uL, 730 uL, 740 uL, 750 uL, 760 uL, 770 uL, 780 uL,790 uL, 800 uL, 810 uL, 820 uL, 830 uL, 840 uL, 850 uL, 860 uL, 870 uL,880 uL, 890 uL, 900 uL, 910 uL, 920 uL, 930 uL, 940 uL, 950 uL, 960 uL,970 uL, 980 uL, 990 uL, and 1000 uL.

Sample channels on the fluidic device can have a volume of less thanabout 1 mL, for example less than about 500 ul, for example less thanabout 200 uL. In some cases, one or more sample channel on the fluidicdevice can have a volume of less than about 10 uL, 20 uL, 30 uL, 40 uL,50 uL, 60 uL, 70 uL, 80 uL, 90 uL, 100 uL, 110 uL, 120 uL, 130 uL, 140uL, 150 uL, 160 uL, 170 uL, 180 uL, 190 uL, 200 uL, 200 uL, 210 uL, 220uL, 230 uL, 240 uL, 250 uL, 260 uL, 270 uL, 280 uL, 290 uL, 300 uL, 310uL, 320 uL, 330 uL, 340 uL, 350 uL, 360 uL, 370 uL, 380 uL, 390 uL, 400uL, 410 uL, 420 uL, 430 uL, 440 uL, 450 uL, 460 uL, 470 uL, 480 uL, 490uL, 500 uL, 510 uL, 520 uL, 530 uL, 540 uL, 550 uL, 560 uL, 570 uL, 580uL, 590 uL, 600 uL, 610 uL, 620 uL, 630 uL, 640 uL, 650 uL, 660 uL, 670uL, 680 uL, 690 uL, 700 uL, 710 uL, 720 uL, 730 uL, 740 uL, 750 uL, 760uL, 770 uL, 780 uL, 790 uL, 800 uL, 810 uL, 820 uL, 830 uL, 840 uL, 850uL, 860 uL, 870 uL, 880 uL, 890 uL, 900 uL, 910 uL, 920 uL, 930 uL, 940uL, 950 uL, 960 uL, 970 uL, 980 uL, 990 uL, or 1000 uL.

Leading electrolyte buffer channels on the fluidic device can have avolume within a range of about 10 uL to about 1 mL. In some cases, oneor more leading electrolyte buffer channel on the fluidic device canhave a volume within a range bounded by any two of the following values:10 uL, 20 uL, 30 uL, 40 uL, 50 uL, 60 uL, 70 uL, 80 uL, 90 uL, 100 uL,110 uL, 120 uL, 130 uL, 140 uL, 150 uL, 160 uL, 170 uL, 180 uL, 190 uL,200 uL, 200 uL, 210 uL, 220 uL, 230 uL, 240 uL, 250 uL, 260 uL, 270 uL,280 uL, 290 uL, 300 uL, 310 uL, 320 uL, 330 uL, 340 uL, 350 uL, 360 uL,370 uL, 380 uL, 390 uL, 400 uL, 410 uL, 420 uL, 430 uL, 440 uL, 450 uL,460 uL, 470 uL, 480 uL, 490 uL, 500 uL, 510 uL, 520 uL, 530 uL, 540 uL,550 uL, 560 uL, 570 uL, 580 uL, 590 uL, 600 uL, 610 uL, 620 uL, 630 uL,640 uL, 650 uL, 660 uL, 670 uL, 680 uL, 690 uL, 700 uL, 710 uL, 720 uL,730 uL, 740 uL, 750 uL, 760 uL, 770 uL, 780 uL, 790 uL, 800 uL, 810 uL,820 uL, 830 uL, 840 uL, 850 uL, 860 uL, 870 uL, 880 uL, 890 uL, 900 uL,910 uL, 920 uL, 930 uL, 940 uL, 950 uL, 960 uL, 970 uL, 980 uL, 990 uL,and 1000 uL.

Leading electrolyte buffer channels on the fluidic device can have avolume of less than about 1 mL, for example less than about 500 ul. Insome cases, one or more leading electrolyte buffer channel on thefluidic device can have a volume of less than about 10 uL, 20 uL, 30 uL,40 uL, 50 uL, 60 uL, 70 uL, 80 uL, 90 uL, 100 uL, 110 uL, 120 uL, 130uL, 140 uL, 150 uL, 160 uL, 170 uL, 180 uL, 190 uL, 200 uL, 200 uL, 210uL, 220 uL, 230 uL, 240 uL, 250 uL, 260 uL, 270 uL, 280 uL, 290 uL, 300uL, 310 uL, 320 uL, 330 uL, 340 uL, 350 uL, 360 uL, 370 uL, 380 uL, 390uL, 400 uL, 410 uL, 420 uL, 430 uL, 440 uL, 450 uL, 460 uL, 470 uL, 480uL, 490 uL, 500 uL, 510 uL, 520 uL, 530 uL, 540 uL, 550 uL, 560 uL, 570uL, 580 uL, 590 uL, 600 uL, 610 uL, 620 uL, 630 uL, 640 uL, 650 uL, 660uL, 670 uL, 680 uL, 690 uL, 700 uL, 710 uL, 720 uL, 730 uL, 740 uL, 750uL, 760 uL, 770 uL, 780 uL, 790 uL, 800 uL, 810 uL, 820 uL, 830 uL, 840uL, 850 uL, 860 uL, 870 uL, 880 uL, 890 uL, 900 uL, 910 uL, 920 uL, 930uL, 940 uL, 950 uL, 960 uL, 970 uL, 980 uL, 990 uL, or 1000 uL.

Elution channels on the fluidic device can have a volume within a rangeof about 10 uL to about 1 mL. In some cases, one or more elution channelon the fluidic device can have a volume within a range bounded by anytwo of the following values: 10 uL, 20 uL, 30 uL, 40 uL, 50 uL, 60 uL,70 uL, 80 uL, 90 uL, 100 uL, 110 uL, 120 uL, 130 uL, 140 uL, 150 uL, 160uL, 170 uL, 180 uL, 190 uL, 200 uL, 200 uL, 210 uL, 220 uL, 230 uL, 240uL, 250 uL, 260 uL, 270 uL, 280 uL, 290 uL, 300 uL, 310 uL, 320 uL, 330uL, 340 uL, 350 uL, 360 uL, 370 uL, 380 uL, 390 uL, 400 uL, 410 uL, 420uL, 430 uL, 440 uL, 450 uL, 460 uL, 470 uL, 480 uL, 490 uL, 500 uL, 510uL, 520 uL, 530 uL, 540 uL, 550 uL, 560 uL, 570 uL, 580 uL, 590 uL, 600uL, 610 uL, 620 uL, 630 uL, 640 uL, 650 uL, 660 uL, 670 uL, 680 uL, 690uL, 700 uL, 710 uL, 720 uL, 730 uL, 740 uL, 750 uL, 760 uL, 770 uL, 780uL, 790 uL, 800 uL, 810 uL, 820 uL, 830 uL, 840 uL, 850 uL, 860 uL, 870uL, 880 uL, 890 uL, 900 uL, 910 uL, 920 uL, 930 uL, 940 uL, 950 uL, 960uL, 970 uL, 980 uL, 990 uL, and 1000 uL.

Elution channels on the fluidic device can have a volume of less thanabout 1 mL, for example less than about 500 ul. In some cases, one ormore elution channel on the fluidic device can have a volume of lessthan about 10 uL, 20 uL, 30 uL, 40 uL, 50 uL, 60 uL, 70 uL, 80 uL, 90uL, 100 uL, 110 uL, 120 uL, 130 uL, 140 uL, 150 uL, 160 uL, 170 uL, 180uL, 190 uL, 200 uL, 200 uL, 210 uL, 220 uL, 230 uL, 240 uL, 250 uL, 260uL, 270 uL, 280 uL, 290 uL, 300 uL, 310 uL, 320 uL, 330 uL, 340 uL, 350uL, 360 uL, 370 uL, 380 uL, 390 uL, 400 uL, 410 uL, 420 uL, 430 uL, 440uL, 450 uL, 460 uL, 470 uL, 480 uL, 490 uL, 500 uL, 510 uL, 520 uL, 530uL, 540 uL, 550 uL, 560 uL, 570 uL, 580 uL, 590 uL, 600 uL, 610 uL, 620uL, 630 uL, 640 uL, 650 uL, 660 uL, 670 uL, 680 uL, 690 uL, 700 uL, 710uL, 720 uL, 730 uL, 740 uL, 750 uL, 760 uL, 770 uL, 780 uL, 790 uL, 800uL, 810 uL, 820 uL, 830 uL, 840 uL, 850 uL, 860 uL, 870 uL, 880 uL, 890uL, 900 uL, 910 uL, 920 uL, 930 uL, 940 uL, 950 uL, 960 uL, 970 uL, 980uL, 990 uL, or 1000 uL.

In some cases, a fluidic device comprises more than one channel. Thechannels may be spaced within the fluidic device at a given density. Insome cases, the edge to edge distance between channels is at least about0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm,1.25 mm, 1.5 mm, 1.75 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or10 mm. In some cases, the edge to edge distance between channels is atmost about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9mm, 1 mm, 1.25 mm, 1.5 mm, 1.75 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm,4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm,9.5 mm, or 10 mm. The density of channels may be defined as a ratio ofthe width of the channels to the space (or distance) between channels.In some cases, the ratio of channel width to distance between channelsis at least about 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1,6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 11:1, 12:1, 13:1, 14:1,15:1, 16:1, 17:1, 18:1, 19:1, or 20:1.

In some cases, the total volume of all channels within a microfluidicdevice (e.g., chip) is 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 175 μL, 200 μL, 225 μL,250 μL, 275 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 600 μL, 700 μL,800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL,19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, or 100 mL. In some cases, thetotal volume of all channels within a microfluidic device (e.g., chip)is at

-   most about 1 microliter (μL), 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60    μL, 70 μL, 80 μL, 90 μL, 100 μL, 150 μL, 175 μL, 200 μL, 225 μL, 250    μL, 275 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 600 μL, 700 μL,    800 μL, 900 μL, 1 milliliter (mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7    mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17    mL, 18 mL, 19 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 50 mL, 55 mL,    60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 85 mL, 90 mL, 95 mL, or 100 mL.

Inlets and/or outlets of a fluidic device can be arranged and spacedsuch that they are compatible with standard fluid handling formats. Forexample, inlets and/or outlets can be spaced to line up with wells on a5″×3.33″ titer plate. A device can comprise eight inlets and/or outlets,spaced to correspond with a standard eight-tip pipettor and/or the eightwells in a dimension of a standard 24-, 48-, or 96-well plate. A devicecan comprise twelve inlets and/or outlets, spaced to correspond with astandard twelve-tip pipettor and/or with the twelve wells in a dimensionof a standard 96-well plate. A device can comprise sixteen inlets and/oroutlets, spaced to correspond with a standard sixteen-tip pipettorand/or with the sixteen wells in a dimension of a standard 384-wellplate. A device can comprise twenty-four inlets and/or outlets, spacedto correspond with a standard twenty-four-tip pipettor and/or with thetwenty-four wells in a dimension of a standard 384-well plate. This canenable easier fluid handling from such plates onto the device, forexample via robotic pipet systems or other multi-pipets.

Devices comprising fluidic elements disclosed herein, such as fluidicand pneumatic channels, ports, reservoirs, capillary barriers and/orfluidic circuits can be used for purposes other than isotachophoresis.For example, application of pressure at external ports or reservoirs canbe used to move liquids through a fluidic circuit. Such devices can becombined with other elements, such as mechanical valves, to control flowof liquids. Compartments separated by barriers (e.g. capillary barriers)can be used to perform biochemical reactions, such as amplificationreactions such as PCR, and reaction product can be moved to anothercompartment separated by barriers for subsequent processing.

Isotachophoresis can be conducted using a benchtop system or basestation. For example, FIG. 13A shows a benchtop system 1300 forconducting sample preparation and isotachophoresis on a fluidic devicecartridge 1301. The fluidic device cartridge can be loaded onto thebenchtop system as shown, and a lid with matching covers and controls1302 can be lowered onto the fluidic device cartridge. The benchtopsystem can also include a control panel 1303 with a user interface(e.g., touch screen) for operation of the system.

The system 1300 can comprise an interface assembly configured to receiveand engage the fluidic device pneumatically, electrically and/orfluidically. The interface assembly can comprise a key or orientationdevice to properly orient the fluidic device 1301 within the instrument1300. The interface assembly also can comprise one or more electrodeswhich, when the fluidic device 1301 is engaged, may be positioned invarious device reservoirs. The interface assembly can further comprisepneumatic lines which, when the device is engaged, may communicate withpneumatic ports in the device 1301 as described herein. The system 1300can include a power supply as described herein to apply current and/orvoltage to the electrodes. The system 1300 can comprise electronics asdescribed herein to measure voltage across various electrodes. Thesystem 1300 can include one or more pumps to supply positive or negativepneumatic pressure to the pneumatic lines as described herein. Thesystem 1300 can comprise a temperature sensor as described herein tomeasure temperature at determined positions in fluidic circuits in thedevice 1301. The system 1300 can comprise an optical assembly comprisingone or more light sources to transmit light to one or more determinedpositions in the fluidic device and to detect light, e.g., fluorescentlight, emitted from the fluidic device, for example, upon excitation offluorescent species in the device 1301 as described herein. The system1300 can include a display as described herein to display operatingparameters of the system, such as voltage, temperature, detected light,engagement of a fluidic device, time, stage of processing. The system1300 can be connected through a communications network, such as theinternet, to a remote server through which operation of the system canbe controlled remotely as described herein.

Negative pneumatic pressure may be applied to pneumatic ports in thedevice in order to load one or more of the buffers or samples into thechannel(s) of the device 1301 as described herein. The system 1300 maybe configured to apply a negative pneumatic pressure within a range ofabout 0 mpsi to about 200 mpsi, for example within a range of about 10mpsi to about 80 mpsi.

FIGS. 59A-59C show drawings of an exemplary benchtop system orinstrument 1300 for conducting isotachophoresis on a fluidic devicecartridge 1301. The fluidic device cartridge 1301 can be loaded onto theinstrument 1300 on a holder 1308 and engaged by manifold 1307. Thesystem may include a cover 1302, for example an articulating manifold,which allows for loading and unloading of the fluidic device cartridge1301 from the instrument 1300. The cover 1302 may optionally facilitatecoupling of the device 1301 to the instrument 1300. The instrument 1300may comprise manifold and/or electrode alignment features 1303 to aid incoupling of a pneumatic manifold 1307 and/or electrodes to the correctlocations on the chip 1301. A USB port 1304 may be provided to allow auser to access data generated by the instrument before, during, or afterone or more ITP runs. The instrument may comprise a display 1305, forexample an LCD screen, as described herein. The instrument mayoptionally comprise a chassis with integrated cooling 1306. Theinstrument may optionally comprise a passive manifold return mechanism1309 which may be used to manually access the chip, for example in theevent of power loss. The instrument may comprise a high-voltage powersupply 1310, a pneumatic pump 1311, a thermal controller (e.g. cooler orheater) 1312, a processor (e.g. a printed circuit board) 1313, a channelcloser 1314, an optical detection system 1315, and/or a triggeringsensor(s) (e.g. an infrared sensor) 1316 for run automation, or anycombination thereof.

FIG. 60 shows an exemplary image which may be displayed to a user toinstruct and guide the user through the reservoir loading process. Forexample, the instrument may display visual and/or color-codedinstructions to the user which indicate which buffers go in whichreservoirs, as well as in which order the buffers should be loaded intothe chip. The chip may be configured to enable an easy right-left orleft-right pipetting scheme to facilitate ease of use by the user. Forexample, the chip may be configured to guide the user, which color-codedinstructions, to load the reservoirs in a left-right manner, startingwith the elution buffer (EB), continuing with the high concentrationelution buffer (EBH), the leading electrolyte buffer (LE), the highconcentration leading electrolyte buffer (LEH), and ending with thetrailing electrolyte buffer (TEH) as shown.

FIG. 61 shows a pneumatic manifold 1307 which may facilitate engagementof the microfluidic chip with the instrument. The pneumatic chipmanifold 1307 may comprise a pneumatic pump or system 1311. Thepneumatic chip manifold 1307 may optionally comprise high voltageelectronics (e.g. power supply 1310). The instrument can comprise amotor that moves the manifold into engagement with the fluidic device.Pressure can maintain fluidic, electrical and pneumatic connections.

FIG. 62 shows a cross-section of a chip 6201 in an instrument 6200. Theinstrument 6200 may be substantially similar to any of the instrumentsdescribed herein (e.g. instrument 1300). The instrument 6200 maycomprise a manifold comprising a gasket and mating holes configured tocouple to the top of the chip 6201 in order to align the chip 6201 withthe instrument 6200. The instrument may further comprise a lowtemperature cooling block 6210 (also referred to herein as a thermalcontroller) and/or an optical detection system 6211 for on-chip nucleicacid quantitation as described herein. The chip 6201 may besubstantially similar to any of the chips described herein (e.g. chip3800). The chip 6201 may for example comprise a sample reservoir 6202,an elution reservoir 6203, an elution buffering reservoir 6204, aleading electrolyte reservoir 6205, a leading electrolyte bufferingreservoir 6206, and a trailing electrolyte reservoir 6207. Theinstrument 6200 may comprise one or more electrodes 6208 as describedherein. The electrodes 6208 may be disposed within the instrument 6200at a fixed height such that they are correctly inserted into the desiredreservoirs when the cover of the instrument 6200 is closed. In someinstances, the electrodes 6208 may enter the corresponding reservoirsevery time the instrument is closed. Electrodes 6208 may for example belocated in the trailing electrolyte reservoir 6207, the leadingelectrolyte buffering reservoir 6205, and/or the leading electrolytebuffering reservoir 6206 such that the electrodes do not directlycontact sample material. The electrodes may be triggered to alter orcontrol the applied electric field in response to feedback from asensor, for example a voltage, current, conductivity, or temperaturesensor as described herein. The chip may optionally comprise one or moreadditional reservoirs 6209 in which additional electrodes 6208 may bedisposed. It will be apparent to one of ordinary skill in the art thatthe location of the electrodes, and the number of electrodes, may bealtered as desired to perform a desired ITP run.

FIG. 63 depicts a vertical manifold motion mechanism comprising amechanical assembly design for motion with alignment andauto-retraction. The drawing depicts a motor with a lead screw that maydrive a mechanism into a position guided by alignment pins. Tensionsprings may automatically retract the mechanism by back-driving themotor in case of power or software failure. FIG. 63 shows a motor 6301with lead screw 6302 which may drive a bracket 6303 into a positionguided by alignment pins 6304. Tension springs 6305 may automaticallyretract the mechanism by back-driving the motor in case of power orsoftware failure. The path of travel may be constrained by the shafts6306 traveling through linear bearings 6307. The vertical manifoldmotion mechanism may be located in the mechanism that controls themotion of the articulating manifold. The vertical manifold motionmechanism may allow the instrument to open itself in the event of anunrecoverable loss of power with a chip in place. This may allow thechip to be removed prior to shipping for service.

FIG. 64 shows a design for a horizontal manifold motion mechanismincluding a mechanical assembly design for horizontal motion using arack and pinion. A motor drives a mechanism using a rack and pinionalong a path constrained by guide rails. The drawing shows a motor 6401attached to a pinion 6402 which may drive the corresponding rack 6403 ona bracket 6404 along a path constrained by guide rails 6405. Thehorizontal manifold motion mechanism may be located on the mechanismthat controls the motion of the articulating manifold. This mayfacilitate movement of the manifold forward and backward.

The benchtop system can comprise pressure controls that provide pressureto handle fluids (e.g., sample, buffer, reagents, enzyme solutions,electrolyte solutions) on a fluidic device. The benchtop system canreceive pressure feedback signals to regulate or control the fluidhandling. Fluid handling can be used to load fluids onto a fluidicdevice (e.g., reagents, buffers, samples). Fluid handling can be used toprime fluids (e.g., reagent solutions) into dry channels on a fluidicdevice. Pressure can be regulated using, for example, solenoid valves.

The benchtop system can comprise electrodes or electrical contacts.Electrodes can be part of an electric circuit and can insert intoreservoirs or other openings on a fluidic device to allow application ofan electric field within the fluidic device by the completed circuit.Electrical contacts can couple to corresponding contacts on a fluidicdevice, for example a fluidic device with integrated electrodes.

The benchtop system can comprise one or more detectors or sensors, suchas optical detectors, reflectance sensors, infrared (IR) detectors,electrical detectors, thermal sensors, flow sensors, and pressuresensors, including detectors described further in this disclosure.Optical detectors can include but are not limited to three-axis pointdetectors, complementary metal-oxide semiconductor (CMOS) detectors,charge-coupled device (CCD) detectors, photodiode light sensors,photoresistors, photomultiplier tubes, and phototransistors. Electricaldetectors can include electrodes or other detectors capable of detectinga voltage, voltage differential, current, charge, or other electricalproperty. Electrical detectors can be used to detect the passage of aband of extracted or purified nucleic acids, for example by detecting achange in conductivity at the interface between trailing electrolytesand leading electrolytes. Thermal sensors can include infrared (IR)sensors, probe temperature sensors, thermistors, negative temperaturecoefficient (NTC) thermistors, resistance temperature detectors (RTDs),thermocouples, semiconductor-based sensors, or the like.

The one or more detectors or sensors can be simultaneously orindependently operated and controlled. In some instances, a singlechannel may have a dedicated sensor, for example a thermal or voltagesensor, which operates independently of other sensors dedicated to otherchannels on the microfluidic device. Feedback from the independentsensor may be used to independently control one or more electric fieldson the device. For example, a sensor may detect a change in voltage overtime within a well as described herein and feedback from that sensor maybe used to control the current within the channel. A second sensor mayact on a second channel in a similar, but independent, manner. In someinstances, a sensor may detect a change in current over time within awell and feedback from that sensor may be used to control the voltagewithin the cannel.

The benchtop system can comprise one or more thermal controllers thatcontrol a temperature on a fluidic device or a part of a fluidic device.Thermal controllers can comprise components including but not limited toresistive heaters, fluid-based heating or cooling systems, and Peltierdevices. Thermal controllers can be fabricated from materials includingbut not limited to metals (e.g., platinum, titanium, copper, gold),carbon, and indium tin oxide (ITO). Thermal controllers can comprisetemperature sensors, which can be used to monitor the temperature beingcontrolled and provide temperature feedback for thermal control. Thermalcontrollers can be used with computer control systems, as discussedfurther in this disclosure. For example, temperature sensors (e.g.,infrared sensors) can be used to monitor a change in temperature inchannels on a chip. Such temperature changes can be indicative of alocation of an ITP band (e.g, a band of nucleic acid) during an ITPprocess, which temperature difference can be due to a change inconductivity between the leading electrolytes and trailing electrolytes.In some cases, thermal controllers are operated without temperaturefeedback.

FIG. 65 depicts a heat pipe with thermoelectric cooler design forkeeping an area at a prescribed temperature remote from the location ofthe thermoelectric cooler. FIG. 65 shows a surface 6501 to be cooled toa specific temperature through a large thermal contact 6502 which maylead to copper heat pipes 6503 and a heat sink 6504. Cooling may beprovided through the thermoelectric cooler 6505 and fan 6506. The copperheat pipe 6503 and auxiliary heat sink 6504 may be configured coolsurface 6501 to a prescribed temperature without the need for thethermoelectric cooler to be directly adjacent to the surface 6501.

FIG. 66 shows another exemplary benchtop instrument 1300 for conductingautomated isotachophoresis and/or sample preparation on a fluidic devicecartridge. FIG. 66 shows a drawing of a beta prototype instrument 1300showing the, location of placement of the fluidic device or chip 1301 tobe controlled. The instrument 1300 may comprise an articulating manifold1302 configured to allow for loading and unloading of fluidic device1301 and coupling of instrument 1300 to device 1301. The instrument 1300may further comprise a strike plate 1303 for electromagnetic eanifoldsealing to the fluidic device 1301.

Techniques of the present disclosure (including, e.g., the use offluidic devices and/or benchtop systems discussed herein) can providequick processing times. For example, a sample comprising nucleic acidscan be prepared (e.g., removal of embedding material, tissue disruption,cell lysis, nucleic acid de-crosslinking) and have nucleic acidsextracted or purified for subsequent analysis, use, or storage.

Detection and Quantitation

Techniques of the present disclosure can employ one or more detectors.Detectors can be integrated into fluidic devices or located externallyto a fluidic device. Detectors can be used for quantitation of nucleicacid in a sample, for example by fluorescent measurement or ultraviolet(UV) radiation (e.g., for measurement of quantity or purity, such as bymeasurement of A260/A280), or for providing a qualitative measure of thenucleic acids in the sample. Nucleic acids can be detected while locatedon a fluidic device, for example while within a purification zone (e.g.,ITP channel) or reservoir (e.g., elution reservoir). The concentrationof the nucleic acids may be detected (or calculated based on a quantitymeasurement in a known volume such as in the elution well as describedherein). Nucleic acids can be labeled, such as with dyes, and thefluorescence intensity of the nucleic acids can be measured by adetector and used to quantify the nucleic acids present (see, e.g., FIG.14). Nucleic acids can be labeled prior to loading on a fluidic device,while in a fluidic device, or after recovery from a fluidic device.

Use of a detector can enable quantitation of nucleic acids from sampleswith a high sensitivity or a low limit of detection. For example,nucleic acids can be detected (e.g., in-line in an isotachophoresischannel) at limit of detection of less than or equal to about 1000picograms per microliter (pg/μL), 100 pg/μL, 10 pg/μL, 1 pg/μL, 0.9pg/μL, 0.8 pg/μL, 0.7 pg/μL, 0.6 pg/μL, 0.5 pg/μL, 0.4 pg/μL, 0.3 pg/μL,0.2 pg/μL, or 0.1 pg/μL. Nucleic acids can be detected (e.g., in-line inan isotachophoresis channel) at a limit of detection of less than orequal to about 1000 picograms (pg), 100 pg, 10 pg, 1 pg, or 0.1 pg.

Use of a detector can enable identification or qualification of nucleicacids in a sample. For example, techniques such as nucleic acidamplification (including, e.g., PCR, real-time PCR, andreverse-transcription PCR), hybridization (including, e.g., fluorescentin situ hybridization (FISH) and Q-FISH), and sequencing can be used toidentify the presence or absence of, and optionally quantify, aparticular sequence within nucleic acids in a sample.

Detectors can be used in the control of nucleic acid extraction orpurification operations. For example, a detector can detect a band ofnucleic acids concentrated by isotachophoresis. When the concentratednucleic acids reach a certain location within the device, the processcan be ended (e.g., electric fields can be turned off) and extracted orpurified sample can be recovered from the device.

Detectors can include but are not limited optical detectors andelectrical detectors, thermal sensors, and pressure sensors (e.g.,pressure transducers). Optical detectors can include but are not limitedto three-axis point detectors, complementary metal-oxide semiconductor(CMOS) detectors, charge-coupled device (CCD) detectors, photodiodelight sensors, photoresistors, photomultiplier tubes, andphototransistors. Optical detection can be achieved by LED illuminationpaired with photodiode detection. Electrical detectors can includeelectrodes or other detectors capable of detecting a voltage, voltagedifferential, current, charge, or other electrical property. Forexample, electrical detectors can be used to detect the passage of aband of extracted or purified nucleic acids.

Nucleic acids can be labeled with one or more nucleic acid dyes, forexample a fluorescent intercalating dye. The nucleic acids may belabeled with one or more of the following intercalating dyes: ethidiumbromide, propidium iodide, DAPI, 7-AAD, YOYO-1, DiYO-1, TOTO-1, DiTO-1,BOBO-1, POPO-1, YO-PRO-1, TO-PRO-1, TO-PRO-3, TO-PRO-5, SYBR® Green I,SYBR® Green II, SYTO™ 9, SYTO™ 13, Quantifluor®, EvaGreen®, PicoGreen®,Acridine Orange, or the like. It will be apparent to one of ordinaryskill in the art that the fluorescent dye may be chosen based on thedesired detection location, detection method, and/or the downstreamprocess(es) which the DNA may be used for following ITP.

FIG. 67 shows a plot of peak area response to nucleic acid mass forvarious nucleic acid binding dyes to assess the possible feasibility ofeach dye for in-line (i.e., on-chip) nucleic acid quantitation duringand/or after ITP. The background-subtracted peak area responses wereplotted as a function of input nucleic acid mass (in ng) and shown on alog-log plot. EvaGreen®, Quantifluor®, Syto™ 13 in sample, and Syto™ 13in sample with added LE was assessed for nucleic acid input masses of0.01 ng, 0.1 ng, 1 ng, 10 ng, 100 ng, and 1000 ng. Only one of thecandidate dyes, Qunatifluor®, demonstrated a linear response below 1 μg(1000 ng) input mass of nucleic acid. Some of the nonlinearilty of theSyto™ 13 response can be explained by dye being stripped off the DNAduring ITP. Adding dye to the LE in addition to the sample recovered thelinear relationship between nucleic acid input mass and peak area.EvaGreen® had both high background and a nonlinear response for allnucleic acid input masses assessed. All of the dyes tested lostlinearity at high input mass (not shown). The preferred embodiments fordye addition to the ITP system for in line quantitation are (1)intercalating dye added to the LE and not the sample (e.g. PicoGreen,Syto 9, Syto 13 or EvaGreen dyes) or (2) dye added to the sample and theLE (e.g. PicoGreen, Syto 9, Syto 13 or EvaGreen dyes), or optionally (3)dye added to sample only (e.g. PicoGreen and EvaGreen dyes).

FIG. 68 shows a plot of peak width response to nucleic acid mass forvarious nucleic acid binding dyes which may, for example, be used toquantify the amount of nucleic acid in the ITP system. Severalcharacteristics of the peak can be measured in order to calculate theamount of nucleic acid passing the detector. Traditional chromatographicmethods consider the peak height and/or area (e.g. as shown in FIG. 67).Assuming the generation of fluorescence is linearly proportional to themass of nucleic acid present, either of these approaches will produce aresponse that is linear with nucleic acid mass. If the underlyingrelationship is not linear, then there will be no fundamentalrelationship between peak area or peak height and nucleic acid mass. Insuch instances, the width of the peak at a fixed signal value can beshown to be linearly proportional to the natural logarithm of nucleicacid mass even when the response is fundamentally non-linear. Data shownin FIG. 68 demonstrate this log-linear relationship between peak widthand nucleic acid input for the dye Syto™ 13 when present in both sampleand LE, or solely in LE. Unlike the peak areas shown in FIG. 67, bothconditions of Syto™ showed a linear relationship between the measuredpeak width and the input mass of the nucleic acids, which may be of usefor on-chip quantification of the mass of the ITP band.

FIGS. 69A-69C depict Quantifluor® incompatibility with PCR. gDNA waspurified using isotachophoresis in the presence and absence of PromegaQuantifluor® at a concentration that was empirically determined toensure linearity of fluorescence intensity up to 5 ug DNA (1× in 225 ultotal sample volume). Recovered eluates were then serially diluted(“df”=dilution factor) and run through qPCR using Qiagen qBiomarker MRefassay (FIG. 69A) to test compatibility with intercalator-based (SYBRGreen I) qPCR and BioRad RPP30 HEX (FIG. 69B) and BioRad RPP30 FAMassays (FIG. 69C) to test compatibility with Taqman-probed based qPCR.Dilutions were made using 10 mM Tris-Cl pH 8.0. Dilution correctedyields from qBiomarker Mref assay were similar for all samples,consistent with a lack of dye-induced inhibition in intercalator-basedqPCR. However, a drop in dilution correct yield was observed withQuantifluor samples in both of the Taqman probe-based qPCR assays,indicating Quantifluor inhibits Taqman probe-based qPCR.

FIG. 70 depicts PicoGreen compatibility with Qubit dsDNA Assay.ThermoFisher Qubit dsDNA Assay is a fluorescence-based DNA quantitationassay that is ubiquitously used upfront of many downstream applications.Purified gDNA at three different concentrations was mixed with PicoGreento a final concentration of 5.7× in a 35 ul eluate volume, the estimatedmaximum concentration needed to ensure linearity in fluorescence across5 ng-5 ug dynamic range of DNA inputs in one implementation of DNApurification through a microfluidic isotachophoretic chip. Samples alongwith no dye controls were quantified using Qubit dsDNA Broad RangeAssay. Comparable quants from samples with and without dye support alack of interference of PicoGreen with the Qubit dsDNA Assay.

FIG. 71 depicts Syto13 compatibility with Qubit dsDNA Assay. PurifiedgDNA at two different concentrations was mixed with Syto13 to a finalconcentration of 5.7× in a total volume of 35 ul, the estimated maximumconcentration needed to ensure linearity in fluorescence across 5 ng-5ug dynamic range of DNA inputs in one implementation of DNA purificationthrough a microfluidic isotachophoretic chip. Samples along with no dyecontrols were quantified using Qubit dsDNA High Sensitivity Assay fromsamples with and without dye support a lack of interference of Syto13with the Qubit dsDNA Assay.

FIGS. 72A-72B depict PicoGreen compatibility with PCR. Purified genomicDNA at varying concentrations of DNA was mixed with PicoGreen at0.0057×, 0.057×, 0.57×, 5.7× and 28.4× or 57× concentrations, each in afinal volume of 100 ul. Samples were run through qPCR using either KAPAhgDNA Quant & QC Assay (FIG. 72A) to test compatibility withintercalator-based (SYBR Green I) qPCR and BioRad RPP30 FAM qPCR (FIG.72B) to test compatibility with Taqman probe-based qPCR. Elevatedbaselines were observed with the 28.4× and 57× reactions resulting indelayed Ct values. However, Ct values for reactions containing 5.7× orless were similar, supporting the compatibility of PicoGreen with bothclasses of qPCR assays up to 5.7× concentration.

FIGS. 73A-73C depicts Syto13 compatibility with PCR. gDNA was purifiedusing isotachophoresis in the presence and absence of Syto13 at aconcentration that was empirically determined to ensure linearity offluorescence intensity up to 5 ug DNA (1× in 225 ul total samplevolume). Recovered eluates were then serially diluted (“df”=dilutionfactor) and run through qPCR using Qiagen qBiomarker MRef assay (FIG.73A) to test compatibility with intercalator-based (SYBR Green I) qPCRand BioRad RPP30 HEX (FIG. 73B) and BioRad RPP30 FAM assays (FIG. 73C)to test compatibility with Taqman-probed based qPCR. Dilutions were madeusing 10 mM Tris-Cl pH 8.0. Dilution corrected yields within each assaywere similar, supporting the compatibility of Syto13 with both classesof qPCR assays.

FIG. 74 depicts PicoGreen compatibility with amplicon-based sequencinglibrary prep. Illumina TruSight Tumor 15 is a multiplexed PCR-basedsequencing library prep assay designed for the targeted enrichment of 15genes commonly mutated in solid tumors. TruSight 15 sequencing librarieswere prepared from gDNA that was mixed with PicoGreen at 1× and 4.4×concentrations in a total volume of 298.5 ul. (4.4× is the estimatedmaximum concentration needed to ensure linearity in fluorescence across5 ng-5 ug dynamic range of DNA inputs in a second implementation of DNApurification through a microfluidic isotachophoretic chip.) A controllibrary (no dye) was also prepped. The three libraries were barcoded andthen sequenced using Illumina sequencing technology, and sequencing datawas downsampled to normalize coverage across the three conditions. Shownare Bland-Altman correlation plots for the two PicoGreen libraries. Alsoshown are statistical metrics for each library. Slight coverage bias ordifferences in top level sequencing metrics were observed betweenlibraries.

FIG. 75 shows Syto13 compatibility with amplicon-based sequencinglibrary prep. gDNA was purified using isotachophoresis in the presenceand absence of Syto13 at a sample concentration empirically determinedto ensure saturation of DNA up to 5 ug. Recovered eluates were then usedto generate TruSight 15 sequencing libraries. Control libraries (no dye)were also prepped. The libraries were barcoded and sequenced usingIllumina sequencing technology. Raw reads were then used to calculatenormalized coverage for each of the 253 targets. Shown is Bland-Altmancorrelation plot. Also shown are statistical metrics for the library. Nobias was observed between libraries.

FIGS. 76A-76B and 78 depict PicoGreen incompatibility with whole genomesequencing library prep. KAPA HyperPlus generates a whole genomesequencing library using fragmentase to enzymatically shear gDNA beforeend-repair and A-tailing the gDNA fragments. The A-tailed genomicfragments are then ligated to sequencing adapters before being PCRamplified and purified using AMPure magnetic bead technology. Shown inFIGS. 76A-76B is fragment length metrics as determined using Agilent'sBioAnalyzer DNA High Sensitivity Assay for whole genome KAPA HyperPluslibraries prepared from gDNA purified using isotachophoresis withoutPicoGreen and with PicoGreen at 4×, 1×, and 0.5× concentrations. Modefragment lengths (left) and gel images (right) show that samplespurified with PicoGreen were longer than control, with the 4× PicoGreensamples being nearly 2× as long as control. Shown in FIG. 78 is libraryquantitation by Qubit dsDNA High Sensitivity Assay. Samples purified inthe presence of PicoGreen exhibited comparable yields as controlsamples.

FIGS. 77A-77B and 79 depict Syto13 compatibility with next generationsequencing library prep. KAPA HyperPlus generates a whole genomesequencing library using fragmentase to enzymatically shear gDNA beforeend-repair and A-tailing the gDNA fragments. The A-tailed genomicfragments are then ligated to sequencing adapters before being PCRamplified and purified using AMPure magnetic bead technology. Shown inFIGS. 77A-77B is fragment length metrics as determined using Agilent'sBioAnalyzer DNA High Sensitivity Assay for whole genome KAPA HyperPluslibraries prepared from gDNA purified using isotachophoresis in thepresence and absence of Syto13. Mode fragment lengths (left) and gelimages (right) show that samples purified with Syto13 were only slightlylonger than both control (no Syto13) and samples purified in thepresence of 1× Syto13. Shown in FIG. 79 is library quantitation by QubitdsDNA High Sensitivity Assay. Samples purified in the presence of Syto13exhibited comparable yields as control samples.

FIGS. 80A-80D depict Syto13 compatibility with whole exome nextgeneration sequencing library prep. Agilent SureSelect XT Technology ishybrid-capture based technology that is designed to target enrichgenomic regions of interest through solution-phase hybridization toultralong cRNA baits. Because whole genome libraries generated with andwithout Syto13 showed a modest difference in fragment length, librarieswere subjected to double sided size selection using AMPure XP beadsfollowed by whole exome target enrichment using SureSelect XT Exome V6.The whole exome libraries were then sequenced using Illumina sequencingtechnology. Raw reads were then used to calculate normalized coveragefor each of the targets. Shown are percent aligned reads, percent Q20high quality aligned reads, and percent aligned with MAPQ>=20 for eachlibrary (FIGS. 80A-80C) and box plots of the mean proportionaldifferences as determined by combinatorial (n=2) Bland-Altman analysis(FIG. 80D). Coverage bias was observed with both the 75% and 100%saturation Syto13 libraries relative to control.

FIG. 81 shows a block diagram that describes an optical signalprocessing algorithm for an optical detector. The raw optical signalacquired by the optical detector may for example be measured asphotodiode current at each lane. Individual current readings mayoptionally be summed by the processor (for example in firmware) in orderto increase the signal to noise ratio (SNR) of the readings before beingrecorded into a run data file. The summed values may be corrected forlane to lane variation in firmware using instrument specific parametersstored in firmware.

In Step 8101, the normalized signal may be recoded into a data file.

In Step 8102, the baseline may be subtracted from the normalized signalin order to approximate and correct for temporal shifts the opticalbaseline. The baseline can represent several imperfections of the systemsuch as DC offset in the analog electronics, auto-fluorescencebackground from the chip and optical components, stray light, error inthe normalization model, and/or temperature effects. Any, a combination,or all of these effects can change as a function of time. However, it isexpected that they should change more slowly than the fluorescent bandpasses the optical detector. Therefore, two techniques may be used aloneor in combination to estimate the baseline of the peak. In the firsttechnique, a median filter with a time window much greater than the peakwidth may be used. Such a technique may provide an easy to implementstrategy which may produce a non-linear baseline. In some instances,however, this first technique may generate a peak which can influencethe baseline value, thereby potentially skewing the data. A secondtechnique for estimating the baseline of the peak includes using alinear regression of data prior to the peak in order to create a linearmodel of the baseline during the peak window. While this technique maybe slightly more difficult to implement than other techniques in atleast some instances, the technique may have the benefit of being morerobust.

In Step 8103, the extent of cross-talk within the chip and/or system maybe corrected for. Due to optical scatter inside the optics sub-assemblyand the within the chip, fluorescence can be recorded at neighboringdetectors from material in other lanes. In order to correct for this,each instrument and/or chip model may have the extent of crosstalkcharacterized. This may allow for the deconvolution of thesimultaneously recorded intensities from all eight channels into the“true” intensities arising from each lane.

At Step 8104, the location of the peak may then be determined byselecting the maximum value within a time window of each channelsoptical signal.

At Step 8105, several characteristics of the peak can be measured inorder to calculate the amount of nucleic acid passing the detector.Traditional chromatographic methods often consider the peak heightand/or area of the recorded fluorescence signal. Assuming the generationof fluorescence is linearly proportional to the mass of nucleic acidpresent, either of these approaches may produce a response that islinear with nucleic acid mass. If the underlying relationship is notlinear, then there is no fundamental relationship between peak area orpeak height and nucleic acid mass. The width of the peak at a fixedsignal value can be shown to be linearly proportional to the naturallogarithm of nucleic acid mass even when the response is fundamentallynon-linear. Any combination of, or all of the three peak characteristics(i.e. width, height, and/or area) may be calculated. If a fundamentalmodel can be constructed either from a linear response to peak area, ora log-linear response to peak width, a calibration model can bedetermined for each instrument and stored in system memory. The modelmay use one or both characteristics to determine the mass of nucleicacid with the lowest uncertainty possible. In the event that nofundamental relationship can be established, calibration data can becollected at high density and stored in system memory as a lookup table.

At Step 8106, linear interpolation between points on the lookup tablecan allow for calculation and estimation of nucleic acid mass across thecharacterized measurement range.

FIG. 82 shows a drawing of a mechanical optical assembly design forillumination and detection of the florescence of a sample bound to adye. This design is an intended sub-assembly in a benchtop controllerinstrument. A mechanical housing 1400 contains optical components that,when a source is applied at the plane 1402, directs the excitation lightthrough the sample plane 1401 and captures the sample fluorescence atthe detection plane 1403.

FIG. 83 shows the optical path that achieves excitation of a samplebound dye fluorescence and the capture of the emitted light from thesample bound dye fluorescence. The source light is shown through aspatial filter 1412 and into a lens 1413 that collimates the light. Thislight then passes through a spectral filter 1410 after which a smallpercentage of that light is reflected by the coated glass 1414 and therest of the light, dominant percentage, passes through the coated glass1414. The reflected light path travels down through a light diffuser1408 and on to the feedback sensor detection plane 1405. This light isused as a feedback loop to help calibrate and normalize the detectionalgorithm in software. The light that passes through continues to asecond glass pain 1416 where it is reflected up to the sample/dye planeafter passing through a lens 1415. Once the light reaches the samplebound dye and excites the dye, light is emitted back through the lens1415 and through a spectral filter 1409. The light then continues on toa lens 1407 where it is focused and passes through a spatial filter 1406onto an electro-optical sensor at the detection plane 1404.

End of Run Triggering

When purifying a sample using ITP, it can be important to accuratelystop applying current when the sample ITP zone is in the elutionlocation (e.g., a channel or a reservoir). The present disclosureprovides techniques for assessing the ITP zone position, which can beused to trigger the end of a purification run. These techniques caninclude measurement of driving voltage, measurement of conductivity, andmeasurement of temperature.

FIG. 15 shows a schematic of an ITP channel 1500, with drivingelectrodes placed in the buffered elution electrode (EH) reservoir 1501and the buffered leading electrolyte (LEH) reservoir 1502, and a groundelectrode placed in the buffered trailing electrolyte (TEH) reservoir1503. Conductivity detector (e.g., capacitively-coupled contactlessconductivity detector (C4D)) electrodes 1504 can be placed outside ofthe chip, such as near the elution reservoir 1505, as shown on the leftside of the figure. The channel can also comprise a leading electrolytereservoir 1506 and a sample reservoir or injection point 1507. Gas portsare indicated by small circles on the far left and right edges of thechannel. Gas ports can be used to automatically load or prime fluidsinto the channels from the attached reservoirs, for example using vacuumor applied pressure.

FIGS. 84A-84F show a control scheme for how the electrical circuitcreated by the electrodes in channel 1500 of FIG. 15 may be verified(i.e. checked for continuity). Electrical continuity may be detectedduring channel priming with the buffers prior to user input of thesample, as shown in FIG. 84A, or after inputting the sample into thechannel 8402 (e.g. via direct injection), as shown in FIGS. 84B-84C.FIG. 84A shows an electrical circuit path 8401 between high-voltageelectrodes located in the buffered elution electrode (EH) reservoir(i.e. 1501 of FIG. 15) and the buffered leading electrolyte (LEH)reservoir (i.e. 1502 of FIG. 15). The circuit being tested does notcross the sample channel 8402, therefore loading in the channel whichdefines path 8401 can be verified prior to sample loading. Testing path8401 before loading the sample may in some instances prevent the userfrom inputting sample into the sample channel 8402 if priming hasfailed. FIG. 84B shows an electrical circuit path 8403 betweenhigh-voltage electrodes located in the buffered elution electrode (EH)reservoir (i.e. 1501 of FIG. 15) and buffered trailing electrolyte (TEH)reservoir (i.e. 1503 of FIG. 15). FIG. 84C shows an electrical circuitpath 8404 between high-voltage electrodes located in the bufferedleading electrolyte (LEH) reservoir (i.e. 1502 of FIG. 15) and bufferedtrailing electrolyte (TEH) reservoir (i.e. 1503 of FIG. 15).

FIG. 84D shows a schematic of a method for detecting if a channel (e.g.channel 1500 of FIG. 15) has electrical continuity. At Step 8411, theelectrode pair of interest may be activated. A source current and sinkcurrent may be set at a pair of electrodes. The sink current may betaken from a grounded electrode that will sink all available current.Alternatively, both electrodes may be actuated by current controlcircuits. This circuit can for example be set to supply at least 10 μA.Alternatively or in combination, voltage control may be used. Thevoltage on the source electrode may for example be set to at least 10 V.Measurements may be taken of the electrode voltage difference (Step8412) and of the total current flowing (Step 8413) along the path. Inthe case of a broken fluidic connection in the microfluidic channel, anelectronic circuit attempting to supply constant current may not be ableto fulfill its target, so a measure of the real current may be done. AtStep 8414, the ratio of the voltage to the current may be taken in orderto determine the channel resistance. At Step 8415, the resistance may besubjected to a threshold to determine channel continuity. In someinstances, the resistance may be checked continuously or nearlycontinuously prior to a timeout in order to enable the current to bedeactivated as soon as the resistance drops below a pre-determinedthreshold value. Alternatively, the resistance may be measuredthroughout a separation, and a failure of continuity may be determinedby detecting a pre-determined number of timepoints are above threshold.At Step 8416, a failure event may be reported to the user and/or thechannel or electrodes may be disabled by the instrument. Shutting downpoorly-loaded channels may prevent damage to adjacent channels and/orthe instrument itself during ITP. FIG. 84E shows an exemplary currenttrace from a successful connection 8417 followed by a failed connection8418 in a single channel. FIG. 84F shows an exemplary voltage trace froma successful connection 8417 followed by a failed connection 8418 in asingle channel.

One method for measuring the position of an ITP band is to measure thevoltage or the resistance of the channel, such as between the drivingelectrode and the ground electrode. In systems with more than twoelectrodes, this measurement may be taken between any pair ofelectrodes. This measurement can be made readily, as the voltage drivingelectrophoresis is also the measurement voltage. Throughout thepurification process, the voltage can increase as the trailing ion fillsthe channel. However, the elution reservoir can have a largecross-section, so the contribution to overall resistance can be small.Hence, changes in the buffer conductivity in this region may notstrongly impact the overall channel resistance, and the voltage can stoprising when the ITP zone enters the elution reservoir. This can be usedas a signal to stop applying current and end the run.

To assess this voltage change, the derivative of voltage can becalculated, for example as shown in FIG. 16. The Lanzcos differentiationmethod can be used to suppress high frequency noise. Thresholds can beset for the derivative, and when the derivative passes the threshold, atrigger is performed. In some cases, introducing additional triggers canimprove the robustness of the control. For example, FIG. 16 shows fourtrigger points. In some cases only two of these triggers are used tochange the driving current (e.g., triggers 1 and 4), while the others(e.g., triggers 2 and 3) are used to mark time points in the run, whichcan improve the timing of trigger 4. FIG. 17 shows derivative analysisof the voltages in FIG. 16, with arrows representing the derivativethresholds used to choose the trigger points.

FIG. 16 shows example data from measuring the driving voltage. Eachvertical line represents a trigger point. The two lines represent twoelectrodes, the electrodes in the EH and LEH reservoirs, with respect tothe ground electrode. Points A, B, C, and D correspond to the time atwhich the ITP zone is in the corresponding location marked in FIG. 15(A, B, C, and D; labeled 1508, 1509, 1510, and 1511, respectively). Insome cases, the conductivity everywhere in the channel can affect theoverall driving voltage, which may make it more difficult to assess whatis happening near the elution reservoir.

A second method for detecting the position of an ITP band is to make alocalized measurement of the conductivity. This can be done using acapacitively coupled contactless conductivity detector (C4D). Thismethod can use high frequency alternating current to pass through thechannel wall and couple to the electrolyte. This localized measurementcan be taken at the elution reservoir itself. This technique can reduceor remove the ambiguity associated with measurements taken over theentire channel. In this technique, the end of run trigger can be chosenas soon as a change is seen in the conductivity at the elution reservoirconductivity detector, for example as shown in FIG. 18.

C4D detection can be performed with electrodes placed below the elutionchannel. Maximizing the electrode area can reduce the necessary drivingfrequency. For example, driving frequencies can be used from about 100kHz to about 10 MHz, with electrode contact pads between about 0.2 mm²and about 50 mm². C4D sensors can be implemented with electricalcomponents including resistors, capacitors, a diode bridge, andhigh-frequency operational amplifiers, with a high frequency signalsource such as from a direct digital synthesizer. FIG. 19 shows anexemplary schematic of a C4D sensor implementation.

A third method for detecting the position of an ITP band is to make alocalized measurement of temperature near the elution reservoir. Thismeasurement can be made with temperature sensors including athermocouple or an infrared temperature sensor. The sensor can be placedunder the channel near the elution reservoir and can monitor thetemperature over time. When the lower-mobility trailing ions displacethe higher-mobility leading ions (e.g. the LE-TE interface of the ITPzone), the electric field in the channel can increase, and thetemperature can rise. During isotachophoresis, lower mobility trailingelectrolyte ions and higher mobility leading electrolyte ions may meetat an isotachophoresis interface. The ITP interface may comprise thesample nucleic acids concentrated between the leading electrolyte ionsand trailing electrolyte ions. A temperature rise can detect thepresence of the ITP interface between the higher-mobility leading ionsand the lower-mobility trailing ions, and thus also indicates thepresence of the nucleic acids therebetween. This temperature rise can be1-10° C.

FIG. 20A and FIG. 20B show exemplary temperature measurement resultsusing a thermal imaging camera. These images show a clear rise intemperature as the trailing ion enters the channel. FIG. 20A shows atemperature map of an ITP channel taken using a thermal imaging camera;the orientation of the channel is the same as in FIG. 15. FIG. 20B showsa plot of temperature over time at the position of Cursor 1 in FIG. 20A.At about 450 seconds, the ITP interface and trailing ion enters theregion, causing an increase in temperature. This temperature rise can bedetected and used as a triggering signal to alter the electric currentapplied to the channel.

The temperature may be measured at a detection location at or near theelution reservoir (e.g. as shown in FIG. 21). In some instances, thedetection location may be located at least about 5 mm from the elutionreservoir. In some instances, the detection location may be located atleast about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm,11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21mm, 22 mm, 23 mm, 24 mm, or 25 mm from the elution reservoir. In someinstances, the detection location may be located at most about 1 mm, 2mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23mm, 24 mm, or 25 mm from the elution reservoir. In some instances, thetemperature sensor may be located at least about 1 mm, 2 mm, 3 mm, 4 mm,5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm,16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, or 25 mmfrom the elution reservoir. In some instances, the temperature sensormay be located at most about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm,19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, or 25 mm from the elutionreservoir.

The temperature sensor may trigger a change in electric current when achange in temperature is sensed. In some instances, the detected changein temperature is within a range of about 0.2° C. to about 5° C. In someinstances, the detected change in temperature is at least about 0.2° C.,0.3° C., 0.4° C., 0.5° C., 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7°C., 8° C., 9° C., or 10° C. In some instances, the detected change intemperature is at most about 0.2° C., 0.3° C., 0.4° C., 0.5° C., 1° C.,2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C.

In some cases, detection of the ITP zone, for example by voltagemonitoring, conductivity measurements, and/or temperature sensing, atone or more trigger points may cause the benchtop controller to alterthe electric current applied to the microfluidic chip. The change may beapplied immediately upon detection or after a pre-determined delay.Detection of the ITP zone may trigger a decrease, increase, or removalof current. For example, detection of the ITP zone at point C 1510 maytrigger a decrease in current in order to increase the residence time ofthe ITP zone in the channel leading to the elution reservoir.Alternatively or in combination, detection of the ITP zone at point D1511 located at or near the elution reservoir may trigger the removal ofelectric current in order to position the ITP zone (and nucleic acids)or a portion thereof within the elution reservoir, well, or region ofthe channel or chip. In some instances, detection of the ITP zone maytrigger a change in electric current after a pre-determined amount oftime. For example, a detection location (for example 1504 or theposition of cursor 1) may be positioned at or near the elution reservoirat a known distance such that the time needed for the ITP zone to travelbetween the detection location and the elution reservoir can becalculated for a given current. The controller may pre-determine atravel time and detection of the ITP zone at the detection location maytrigger a delayed removal of the current after the pre-determined amountof time. In some instances—detecting the ITP zone at a specificdetection location may offer a space-time relationship of the ITP zonewhich may result in more precise triggering than other sensing methods.

In some cases, detection of the ITP zone at a trigger point may causethe electric current applied to the microfluidic chip to changedirections or paths. For example, the electric current may be triggeredto reverse such that the ITP zone reverses direction of travel withinthe channel. In another example, the system may be triggered to stopapplying current between a first pair of electrodes and begin applyingcurrent to a second pair of electrodes to drive the flow of ions along adifferent path. For example, a channel may be “y-shaped” with a firstchannel leading into two side channels which split from the firstchannel at different directions. Current may initially be driven betweenfirst and second electrodes connected to the first channel and a firstside channel, respectively. Without interruption of current, the ITPzone may travel from the first channel to the first side channel.Detection of the ITP zone at a connection between a first channel andtwo side channels may trigger the first and second electrodes to stopdriving current and third and fourth electrodes connected to the firstchannel and a second side channel, respectively, to begin drivingcurrent. The ITP zone will then travel from the first channel to thesecond side channel. In some cases, the first and third electrodes arethe same electrode. In this way, the trigger may cause the current tochange such that the path of the ITP zone changes along the channel.

In some cases, the position of an ITP band may be detected using anycombination of the detection methods described herein.

In some embodiments, the position of an ITP band may be detected bymeasuring the voltage or resistance of the channel in combination withmaking a localized measurement of temperature near the elutionreservoir. The voltage may be measured as described herein. For example,the voltage may be measured during the entire ITP run. In some cases,the voltage may be monitored and changes in voltage, or in thederivative of the voltage, may be used as triggers to change the drivingcurrent as described herein. Alternatively or in combination, changes inthe voltage, or in the derivative of the voltage, may be used to marktime points in the run in order to improve the timing of later triggersas described herein. For example, a first voltage change may indicatethat the ITP band has reached the capillary barrier between the samplechannel and the leading electrolyte channel, which may be used to mark atime point in the run and improve triggering overall. A second voltagechange may indicate that the ITP band has reached the narrowing (i.e.constriction) portion of the LE channel shown in FIG. 40. The secondvoltage change may trigger a change in the driving current after apre-determined amount of time. For example, the second voltage changemay trigger the driving current to switch from the electrodes in theleading electrolyte buffering well and the trailing electrolyte well (asshown in FIG. 84C) to the electrodes in the elution buffering well andthe trailing electrolyte well (as shown in FIG. 84B). The electrode inthe leading electrolyte buffering reservoir may be turned off after thepre-determined amount of time. Alternatively, the polarity of theelectrode in the leading electrolyte buffering reservoir may be reversedafter the pre-determined amount of time. In some embodiments, thepre-determined amount of time may be configured to coincide with the ITPband reaching the integrated quantitation region shown in FIG. 40. Afterswitching the driving current between electrode pairs, the temperaturemay be measured as described herein. For example, an infraredtemperature sensor can be placed under the channel near the elutionreservoir as described herein and as shown in FIG. 40. The temperaturesensor may be configured to monitor the temperature at the sensor overtime as described herein. Temperature differences may be generatedbetween the ions at the ITP interface due to the electric field appliedto the channel and the ionic strength of the ions. The ITP interface maycomprise the sample nucleic acids concentrated between the leadingelectrolyte ions and trailing electrolyte ions. A temperature rise candetect the presence of the ITP interface between the higher-mobilityleading ions and the lower-mobility trailing ions, and thus alsoindicates the presence of the nucleic acids therebetween. Upon detectionof a temperature rise with the temperature sensor (e.g. as shown inFIGS. 91A-91B), the electrical current may be removed after apre-determined amount of time (which may correspond to the distancebetween the IR sensor and the elution reservoir) as described herein.The purified nucleic acids may then be eluted from the elution reservoirand optionally used for further processing as described herein.

In some embodiments, the current applied across the fluidic device maygenerate a first temperature difference at an interface between thenucleic acid analyte in the ITP band and the trailing electrolyte. Thetrailing electrolyte may be warmer than the nucleic acids in the ITPband due to their lower ionic strength. The current applied across thefluidic device may generate a second temperature difference at aninterface between the nucleic acid analyte in the ITP band and theleading electrolyte. The leading electrolyte may be cooler than thenucleic acids in the ITP band. The temperature sensor may be configuredto detect the temperature difference between the cooler leadingelectrolyte and the ITP band, and subsequently detect the temperaturedifference between the ITP band and the warmer trailing electrolyte.

In at least some instances, the current applied across the fluidicdevice may generate a first temperature at the interface between theanalyte and the trailing electrolyte. The current applied across thefluidic device may generate a second temperature an interface betweenthe analyte and the leading electrolyte. The current may be appliedacross the fluidic device such that the temperature difference betweenthe first and second temperatures is sufficient to generate a thermaleffect therebetween (e.g. at a sufficiently high current to generate athermal effect). When the current is stopped within the channel underthe elution reservoir (e.g. at the aperture between the two), thisthermal effect may be sufficient to create a buoyant effect on the ITPband and facilitate entry of the nucleic acids of the ITP into theelution reservoir via the aperture.

Further Processing and Use of Purified Samples

Extracted or purified nucleic acids can be used for sequencing,genotyping, analysis of mutations or polymorphisms, analysis of geneexpression levels, disease diagnosis, disease prediction, cytologicalclassification, paternity or genealogical analysis, or indication ofsuggested treatment modalities.

In preferred embodiments, the extracted or purified nucleic acids (e.g.DNA, RNA) can be used in amplification reactions such as PCT reactions.In some cases, extracted or purified nucleic acids can be used inamplification reactions, including but not limited to loop-mediatedisothermal amplification (LAMP), strand displacement amplification(SDA), helicase-dependent amplification (HDA), rolling circleamplification (RCA), nicking enzyme amplification reaction (NEAR), PCR,reverse transcription PCR, real-time PCR, quantitative PCR (qPCR),digital PCR, and methylation-specific PCR.

Extracted or purified nucleic acids can be used in sequencing reactions,including Maxam-Gilbert sequencing, chain termination sequencing (e.g.,Sanger sequencing), shotgun sequencing, pyrosequencing, bridge PCR,colony sequencing, polony sequencing, sequencing by synthesis, ionsemiconductor sequencing, nanopore sequencing, nanoball sequencing,sequencing by ligation, sequencing by hybridization, and single moleculereal-time sequencing.

Extracted or purified nucleic acids can be used in protein bindingassays, such as DNA footprinting assays. For example, DNase (e.g., DNaseI) can be used to randomly cut DNA molecules of interest. The techniquesof the present disclosure can be used to separate digested DNA from theDNase enzymes, preventing further digestion. In some cases, DNasedigestion can be performed off of a fluidic device, and then the samplecan be loaded onto a fluidic device for purification. In other cases,DNase digestion can be performed on a fluidic device, and once digestionis performed, the nucleic acids can be purified on the fluidic device.

Samples, such as fixed or embedded samples (e.g., FFPE samples), can beused for longitudinal studies, genome-wide association studies, andother large-scale analysis across populations.

Vertical or Column ITP

Planar ITP device designs, such as discussed herein, can utilizehorizontal space for ITP bands to travel. To process samples at highthroughput, such as in the 96-well plate format, it can be advantageousto fit an entire ITP separation system for a sample in a givenfootprint, such as 9 mm×9 mm footprint. One way of doing this is toincrease the height of the system to accommodate more sample volume.This can provide the option to increase total sample volumes into themilliliter range and still process samples with reasonable run times.

In some cases, it can be important to reduce or prevent gravity-drivenflow and/or buoyant flow through such a system. It can also be importantto assemble the electrolyte zones needed for ITP without mixing theelectrolytes.

A vertical or column ITP system can comprise several ITP stages, whereeach stage comprises a column (e.g., plastic) with gel (e.g., agarose)or similar material at the bottom. The gel can have high electrolyticconductivity. Each stage can be prepared by introducing an electrolyteon top of the gel. The gel can slow or prevent liquid flow. To createthe column, the stages can be stacked with the trailing electrolyte atthe top and the leading electrolyte at the bottom. Current can then bedriven through the system. Purified analyte can be recovered byde-stacking the columns and pipetting out.

FIG. 22A shows an exemplary schematic of a vertical (or column) ITPsetup. The vertical ITP device may comprise a column having a pluralityof gel plugs disposed within an interior channel thereof. The gel plugsmay be separated by spaces configured to receive one or more of thebuffers or sample fluids described herein. Each gel plug and space maymake up a stage of the vertical ITP column. The gel plugs in each stagemay comprise a gel material that can support the weight of the water(e.g., aqueous electrolyte solution or sample volume) added to the spaceabove the gel plug. Each gel plug may support a free solution disposedabove it. The vertical ITP device may for example comprise five stages.The first stage may comprise a trailing electrolyte buffer and a firstgel plug configured to support the trailing electrolyte buffer. Thesecond stage, located below the first stage, may comprise a sample oranalyte as described herein and a second gel plug. The third stage,located below the second stage, may comprise a leading electrolytebuffer and a third gel plug. The fourth stage, located below the thirdstage, may comprise an elution buffer and a fourth gel plug. The fifthstage, located below the fourth stage, may comprise a high concentrationleading electrolyte buffer and fifth gel plug. When an electrical fieldis applied to the vertical ITP device, the analyte may migrate in thedirection of gravity from the second stage, through the second gel plug,into the third stage (leading electrolyte buffer), through the third gelplug, and down into the fourth stage (elution buffer). The analyte maycompact into an ITP band as described herein with respect to ITPperformed within a channel. The cross-sectional area of the ITP columncan be approximately 9 mm×9 mm. Such a system may be configured toprocess a sample with an approximate cross sectional column area of 9mm×9 mm. The design can be scaled-up as desired by one of ordinary skillin the art, for example to 96 samples (columns), with overall devicedimensions conforming to a standard microtiter plate. FIG. 22B shows anexemplary image of a vertical ITP set up with a DNA ITP band. The stagesare: Trailing Electrolyte High (TEH), Sample, Leading Electrolyte (LE)and Leading Electrolyte High (LEH). The ITP zone is moving downwardthrough the system. This image does not show the elution stage (E, shownin FIG. 22A) which is the final destination of the analyte.

Computer Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 13B shows acomputer system 1304 that is programmed or otherwise configured tocontrol sample preparation, sample extraction or purification, ordetection. The computer system 1304 can regulate various aspects ofextraction, purification, and detection processes of the presentdisclosure, such as, for example, application of pressure or electricfields, thermal control, detection, quantitation, feedback, andbeginning or ending a process. The computer system 1304 can be anelectronic device of a user or a computer system that is remotelylocated with respect to the electronic device. The electronic device canbe a mobile electronic device.

The computer system 1304 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1305, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1304 also includes memory or memorylocation 1310 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1315 (e.g., hard disk), communicationinterface 1320 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1325, such as cache, othermemory, data storage and/or electronic display adapters. The memory1310, storage unit 1315, interface 1320 and peripheral devices 1325 arein communication with the CPU 1305 through a communication bus (solidlines), such as a motherboard. The storage unit 1315 can be a datastorage unit (or data repository) for storing data. The computer system1304 can be operatively coupled to a computer network (“network”) 1330with the aid of the communication interface 1320. The network 1330 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1330 insome cases is a telecommunication and/or data network. The network 1330can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1330, in some cases withthe aid of the computer system 1304, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1304 tobehave as a client or a server.

The CPU 1305 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1310. The instructionscan be directed to the CPU 1305, which can subsequently program orotherwise configure the CPU 1305 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1305 can includefetch, decode, execute, and writeback.

The CPU 1305 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1304 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1315 can store files, such as drivers, libraries andsaved programs. The storage unit 1315 can store user data, e.g., userpreferences and user programs. The computer system 1304 in some casescan include one or more additional data storage units that are externalto the computer system 1304, such as located on a remote server that isin communication with the computer system 1304 through an intranet orthe Internet.

The computer system 1304 can communicate with one or more remotecomputer systems through the network 1330. For instance, the computersystem 1304 can communicate with a remote computer system of a user.Examples of remote computer systems include personal computers (e.g.,portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® GalaxyTab), telephones, Smart phones (e.g., Apple® iPhone, Android-enableddevice, Blackberry®), or personal digital assistants. The user canaccess the computer system 1304 via the network 1330.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1304, such as, for example, on thememory 1310 or electronic storage unit 1315. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1305. In some cases, thecode can be retrieved from the storage unit 1315 and stored on thememory 1310 for ready access by the processor 1305. In some situations,the electronic storage unit 1315 can be precluded, andmachine-executable instructions are stored on memory 1310.

The code can be pre-compiled and configured for use with a machine havea processor adapted to execute the code, or can be compiled duringruntime. The code can be supplied in a programming language that can beselected to enable the code to execute in a pre-compiled or as-compiledfashion.

Aspects of the systems and methods provided herein, such as the computersystem 1304, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such memory (e.g., read-only memory, random-access memory,flash memory) or a hard disk. “Storage” type media can include any orall of the tangible memory of the computers, processors or the like, orassociated modules thereof, such as various semiconductor memories, tapedrives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All or portions of thesoftware may at times be communicated through the Internet or variousother telecommunication networks. Such communications, for example, mayenable loading of the software from one computer or processor intoanother, for example, from a management server or host computer into thecomputer platform of an application server. Thus, another type of mediathat may bear the software elements includes optical, electrical andelectromagnetic waves, such as used across physical interfaces betweenlocal devices, through wired and optical landline networks and overvarious air-links. The physical elements that carry such waves, such aswired or wireless links, optical links or the like, also may beconsidered as media bearing the software. As used herein, unlessrestricted to non-transitory, tangible “storage” media, terms such ascomputer or machine “readable medium” refer to any medium thatparticipates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1304 can include or be in communication with anelectronic display 535 that comprises a user interface (UI) 1340 forproviding, for example, operational parameters (e.g., processing time,temperature, field strength), nucleic acid quantitation information, orother information. Examples of UI's include, without limitation, agraphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1305. Thealgorithm can, for example, regulate thermal controllers, calculatenucleic acid quantitation, control process functions, and begin or end aprocess.

Kits

This disclosure provides kits useful for conducting isotachophoresisprocesses. Generally, such kits comprise a microfluidic device providedherein and one or more buffers. The buffers may include one or moresample buffers, one or more leading electrolyte buffers, one or moretrailing electrolyte buffers, one or more lysis buffers and/or one ormore elution buffers, in any combination. In some cases, the kit may ininclude one or more enzymes (e.g., RNase, DNAs, nucleases, proteases,proteinases, polymerase). The buffers may be supplied in separate tubes.In some cases, the microfluidic device is pre-loaded with one or morebuffers. The kits may include a set of instructions for operating thedevice and/or processing a sample.

EXAMPLES Example 1—DNA Extraction from FFPE Samples

An FFPE sample from a human patient is obtained. A 1.1× aqueous alkalinebuffer solution (Solution A1) is prepared with 80 mM NaOH, 11 mM DTT,and 0.5% v/v Igepal CA-630 in nuclease-free distilled or deionizedwater. A 10× quenching solution (Solution A2) is prepared with 776 mMHCl and 100 mM Tris base or Trizma base in nuclease-free distilled ordeionized water. Commercially available Proteinase K solutions andRNases are also provided. Alternatively, a neutrally-buffered (e.g., pHfrom about 7.0 to about 8.0) 5-50 mM Tris-HCl solution with 0-80 mMNaCl, 5-10 mM DTT, and 0.1-0.5% v/v IGEPAL CA-630 can be prepared innuclease-free distilled or deionized water.

An FFPE section or scroll is added to a 1.5-2.0 mL microcentrifuge tube.175 μL of Solution A1 is added to the tube. The tube contents areincubated for 1-20 minutes at 50-99.9° C. (in some cases, the tubecontents are incubated for 5-20 minutes at 95-99.9° C.) to deparaffinizethe sample. 20 of Solution A2 are added to the tube to quench SolutionA1 and achieve a buffered solution with pH of about 7-8.25.Alternatively, an FFPE section or scroll can be incubated in 195 μL ofquenched or neutral buffer (e.g., pH from about 7.0 to about 8.0) for1-30 minutes at 50-80° C. to deparaffinize the sample. Otherdeparaffinization protocols that can be used include (1) treating thesample with xylene, followed by one or more washes with 96%-100% ethanolat room temperature, followed by drying of the tissue; (2) incubatingthe sample at an elevated temperature (e.g., 50-100° C.) for 1-30minutes in a buffered aqueous solution at about pH 7 to about pH 8.25;(3) incubating the sample at an elevated temperature (e.g., 50-100° C.)for 1-30 minutes in an alkaline aqueous solution followed by quenchingto a buffered solution with pH of about 7 to about 8.25; or (4)incubating the sample at an elevated temperature (e.g., 50-100° C.) for1-30 minutes in mineral oil.

5 μL of Proteinase K solution is added to the deparaffinized samplesolution to a final concentration of 400-1000 μg/mL (typically 600-700μg/mL) and a final volume of 200 μL. The solution is then incubated for15-60 minutes at about 56° C. Optionally, the solution is furtherincubated for 2-60 minutes at 80-90° C. Optionally, 3 μL of RNase A (orabout 50-200 μg/mL RNase A) is added to the solution. The solution isthen cooled to room temperature, and the FFPE lysate is loaded onto afluidic device for further processing, such as by isotachophoresis(ITP).

Example 2—Comparison of DNA Extraction Yields

DNA was extracted using a bench top controller device to automateisotachophoresis in a fluidic device from (i) qPCR buffer as a post-PCRclean-up (FIG. 3, triangle data points), and (ii) cell culture lysate(FIG. 3, square data points), with yield calculated using qPCR.Published DNA yield data using a traditional solid-phase extractioncolumn (SPE; FIG. 3, diamond data points) are provided for comparison.FIG. 3 shows DNA yield versus input DNA mass. The leading electrolytebuffer used for isotachophoresis comprised 88 mM Tris with 44 mM HCl.Trailing electrolyte was loaded into the trailing electrolyte reservoirand comprised 1.2M Tris with 0.3 M Caproic Acid and 0.6 M MOPS. Thecellular lysate sample was prepared in a second leading electrolytebuffer (sample buffer) comprising 10 mM Tris with 5.6 mM HCl. Extractionof DNA from human Jurkat cell culture lysate was performed at yieldsfrom about 60% to about 90% for input DNA masses from about 10⁻²nanograms (ng) to about 10³ ng. Cells were lysed in an aqueous solutioncomprising 40 mM NaOH for 1 minute and subsequently quenched at a 1:1volume ratio with a buffered acidic solution to bring the final celllysate sample to 10 mM Tris with 5.6 mM HCl and 20 mM NaCl at pH 8.Proteinase K was added to a final concentration of 400 μg/ml within thecell lysate sample volume and incubated for 10 minutes and 56° C. Thelysed sample was then brought to room temperature and loaded onto thefluidic device for isotachophoresis. Extraction of genomic DNA(pre-purified from human Jurkat cells using a commercial SPE kit) spikedinto a buffer comprising 10 mM Tris-HCl pH 8 was performed at yieldsfrom about 90% to about 100% for input DNA masses from about 10⁻¹ ng toabout 10³ ng.

Compared to traditional SPE column kits, the isotachophoresis method anddevice used here allowed for higher yields. This may have been due to ahigher off-chip lysis efficiency with the indicated lysis chemistryfollowed by a more efficient recovery of nucleic acids usingisotachophoresis. The isotachophoresis methods and devices describedherein may provide lower adsorption of nucleic acids samples to thesurfaces of the chip compared to a standard column and/or lower deadvolumes within the fluidic device than a column. The isotachophoresismethods and devices described herein may enable less biased or unbiasedrecovery of nucleic acids based on length and/or sequence, which mayalso provide for higher efficiency recovery. The spiked-in genomic DNAsample performed had a very high recovery (yield) which may indicatethat isotachophoresis has very little systematic loss of sample due tothe isotachophoresis process itself (whereas the cell lysate sample mayhave other factors which contribute to loss of efficiency such as thelysis chemistries used which may be improved for higher yields).

Example 3—Separation of Crosslinked and Non-Crosslinked Nucleic Acids

A deparaffinized and lysed mouse FFPE tissue sample (processed asdescribed in Example 1) comprising crosslinked and non-crosslinkednucleic acids was loaded onto a fluidic device for isotachophoresis withleading electrolyte and trailing electrolyte. The sample was lysed asdescribed in Example 1 and prepared in a leading electrolyte solution toa final concentration of 10 mM Tris with 5.6 mM HCl. The leadingelectrolyte comprised 140 mM Tris with 70 mM HCl. The trailingelectrolyte comprised a mixture of 2.1 M Tris with 0.5 M caproic acid asa spacer ion with a higher effective mobility magnitude than HEPES and0.7 M HEPES as an ion with a lower effective mobility magnitude. Duringisotachophoresis, non-crosslinked nucleic acids, having a highereffective mobility magnitude, focus ahead of the caproic acid zone andbehind the leading electrolyte zone. Crosslinked nucleic acids andsample contaminants focus behind the caproic acid zone, and either aheadof or within the HEPES zone depending on the degree of crosslinking andtheir effective mobility magnitude. FIG. 23 shows two images of DNAseparation in an isotachophoresis channel subsequent to a two hour(upper) or an overnight (lower) digestion to remove crosslinkingproteins from the DNA. Proteinase K was added to the deparaffinizedlysed tissue (quenched to pH 8.2) at final concentration of 700 μg/mlfor digestion. Crosslinked DNA appears at the left end of the channel,separated by spacer ions from the amplifiable non-crosslinked DNA at theright end of the channel. The graph in FIG. 23 shows intensity of DNAsignal versus position in the channel for the two hour 2301 andovernight 2302 digestions.

Example 4—Extraction and Purification of DNA from Lung and Liver FFPESamples

Formalin-fixed paraffin-embedded (FFPE) mouse lung and liver sampleswere obtained (e.g., Zyagen). Seven pairs of FFPE sections (1 cm by 1 cmby 5-10 μm) were processed, with one section from each pair processed byon-device isotachophoresis and one section processed by a differentmethod (Promega ReliaPrep FFPE DNA kit) for comparison. The leadingelectrolyte buffer used for isotachophoresis comprised 88 mM Tris with44 mM HCl. Trailing electrolyte was loaded into the trailing electrolytereservoir and comprised 1.2M Tris with 0.3 M Caproic Acid and 0.6 MMOPS. Samples were deparaffinized by incubation in a 10 mM Tris-HClbuffer with 10 mM DTT, 72 mM NaCl and 0.5% IGEPAL CA-630 at pH 8.0 forapproximately 1 minute at 80° C., and subsequently treated withproteinase K in the same solution for 60 minutes at 56° C. The digestedsample was then incubated for 15 minutes at 90° C. ITP was conducted onone sample from each pair of sections by dispensing 200 μL ofpre-processed sample mixture, including embedding and FFPE tissuedebris, into the sample inlet of a fluidic device. The other sectionfrom each pair was extracted using Promega's ReliaPrep FFPE gDNA kitaccording to manufacturer's protocol. FIG. 24A shows an image of twoneighboring ITP channels on a fluidic device with DNA from FFPE samples,labeled with intercalating dye for visualization. Extracted DNA from thesamples was quantified with qPCR. FIG. 24B shows the quantifiedextracted DNA in nanograms (ng) for each of the seven sample pairs. Foreach pair, the darker left-hand bar shows results for ITP and thelighter right-hand bar shows results for the ReliaPrep kit. The leftmosttwo sample pairs are human liver samples, and the remaining five samplepairs are human lung samples. For all seven sample pairs, the amount ofamplifiable nucleic acids extracted via ITP is significantly higher(typically about 1.5 to 8 times higher amplifiable yields) than theamount of nucleic acids extracted by the ReliaPrep kit.

Example 5—ITP-Based Quantitation of Nucleic Acids

Quantitation of nucleic acids using ITP was tested and compared to qPCR.The comparison was performed over the full range of sample amounts usingan RNaseP human reference gene assay (ABI). Standard or calibrationcurves were generated from 50 qPCR runs (10 replicates each at 5 ordersof magnitude concentrations) and were used to quantify qPCR measurementuncertainty for this range of DNA amounts.

DNA was extracted from 4 million Jurkat cells using a standard kit(e.g., Invitrogen PureLink Genomic DNA kit). For on-device ITP, Jurkatcells were lysed off-chip using a pH 12.7 NaOH solution for 2 minutes,quenched to buffered solution at pH 7.5-8 using a solution ofhydrochloric acid and Tris base, and then treated with Proteinase K forat pH 8 and 56° C. for 10 minutes.

Pre-purified DNA was processed via ITP and quantified in the ITP channelvia fluorescent intensity. The leading electrolyte buffer used forisotachophoresis comprised 88 mM Tris with 44 mM HCl. Trailingelectrolyte was loaded into the trailing electrolyte reservoir andcomprised 1.2M Tris with 0.3 M Caproic Acid and 0.6 M MOPS. The samplewas prepared in a leading electrolyte buffer (sample buffer) comprising10 mM Tris with 5.6 mM HCl. FIG. 14 shows a titration curve of measuredfluorescent intensity from DNA compared to known DNA sample mass, and islinear over seven orders of magnitude, from 0.4 picograms (pg) to about10⁶ pg. The upper left inset of FIG. 14 shows an image of 250 ng of DNAin an ITP channel. The lower right inset of FIG. 14 shows point detectorfluorescent signal intensity on a logarithmic scale for ITP-extractedDNA in an ITP channel, at 250 pg, 2.5 ng, 25 ng, and 250 ng of DNA.

Example 6—Lack of Bias in ITP Extraction and Purification

Mixtures of synthetic 100 base labeled DNA oligonucleotides with 63% A-Tcontent (37% G-C content, HEX label) and DNA oligonucleotides with 68%G-C content (FAM label) were prepared at three concentrations (1 ng/μL,square data points; 10 ng/μL, diamond data points; 100 ng/μL, triangledata points) and five concentration ratios (overall GC- to AT-rich ratiofrom 0.1 to 10). Ratios were calculated from fluorescence plate readermeasurements obtained pre- and post-processing. FIG. 4A shows acomparison of the output GC- to AT-rich ratio versus the input GC- toAT-rich ratio for ITP processing, demonstrating a lack of bias in theITP process.

A mixture of oligonucleotides from a 1 kb DNA ladder (New EnglandBiosciences) was measured for length before and after processing, usingintegrated signals of electropherogram peaks from the Experion 12k DNAanalysis kit (BioRad). Size distribution within the sample before andafter processing was compared for on-device ITP (FIG. 4B, top), QiagenQiaAmp column kit (FIG. 4B, middle), and Invitrogen PureLink column kit(FIG. 4B, bottom). The leading electrolyte buffer used forisotachophoresis comprised 88 mM Tris with 44 mM HCl. Trailingelectrolyte was loaded into the trailing electrolyte reservoir andcomprised 1.2M Tris with 0.3 M Caproic Acid and 0.6 M MOPS. The samplewas prepared in a leading electrolyte buffer (sample buffer) comprising10 mM Tris with 5.6 mM HCl. For each comparison, the top row shows thesize distribution in the recovered output fraction and the bottom rowshows the initial size distribution in the sample.

Example 7—Off- or On-Chip Proteinase K Digestion of Cell Lysate NucleicAcids

FIG. 25A shows an image of a single channel ITP chip loaded with nucleicacid (RNA extraction and proteolytic digest from human cells) stainedwith dye for visualization. On the left 2501 is an ITP band from sampleprocessed off-chip prior to loading with 200 μg/mL proteinase K insample buffer, while on the right 2502 is a sample not processed withproteinase K. The leading electrolyte buffer used for isotachophoresiscomprised 100 mM Tris with 50 mM HCl. Trailing electrolyte was loadedinto the trailing electrolyte reservoir and comprised 1.8M Tris with 1 MCaproic Acid and 1 M MOPS. The sample was prepared in a leadingelectrolyte buffer (sample buffer) comprising 10 mM Tris with 5.6 mMHCl. FIG. 25B shows an image of a single channel ITP chip loaded withnucleic acid (RNA extraction and proteolytic digest from human cells)stained with dye for visualization. On the left 2501 is an ITP band fromsample processed on-chip 200 μg/mL proteinase K in leading electrolyte,while on the right 2502 is a sample not processed with proteinase K. Inboth cases, the sample processed with proteinase K exhibits a tighterITP band, representing nucleic acids not associated with protein (highereffective mobility magnitude), while the sample not processed withproteinase K exhibits a smeared band, representing nucleic acidassociated with variable amounts of protein (lower effective mobilitymagnitude).

Example 8—RNA Extraction from Human Cells Using Off-Chip Lysis andOn-Chip ITP Purification

FIG. 26A shows an image of an RNA ITP band 2601 in a chip channel duringextraction and purification of RNA from cell lysate (Jurkat cells) withDNA digested. FIG. 26B shows an image of a total nucleic acid ITP band2602 in a chip channel during extraction and purification of RNA fromcell lysate (Jurkat cells) without DNA digested. The leading electrolytebuffer used for isotachophoresis comprised 100 mM Tris with 50 mM HCl.Trailing electrolyte was loaded into the trailing electrolyte reservoirand comprised 1.8M Tris with 1 M Caproic Acid and 1 M MOPS. The samplewas prepared in a leading electrolyte buffer (sample buffer) comprising10 mM Tris with 5.6 mM HCl. FIG. 26C and FIG. 26D show graphs of RNAquality electropherograms (measured using the BioRad Experion) for thesamples shown in FIG. 26A and FIG. 26B, respectively. Cell lysis andDNase digestion were performed in a buffered solution at pH 8 containing7M urea, 2M thiourea, and a non-ionic surfactant as discussed herein.These results demonstrate the preparation of high quality RNA with orwithout DNA digestion.

Example 9—Extraction of Whole Lysed Blood Using ITP and 200 μl ChipDevice

FIG. 27A shows results of DNA yield (ng) for ITP (square) compared tocolumn (diamond, Qiagen QiaAmp) extraction of whole mouse blood as afunction of percent by volume of whole blood in starting sample. FIG.27B shows an image of total nucleic acid in an ITP band 2401 during ITPpurification of lysed whole mouse blood on a chip. The leadingelectrolyte buffer used for isotachophoresis comprised 260 mM Tris with130 mM HCl. Trailing electrolyte was loaded into the trailingelectrolyte reservoir and comprised 2.1 M Tris with 0.5 M Caproic Acidand 0.7 M MOPS. The sample was prepared in a leading electrolyte buffer(sample buffer) comprising 10 mM Tris with 5.6 mM HCl.

FIG. 27C and FIG. 27D show white light and fluorescence overlay imagesof ITP chip channels showing physical separation of heme from the bloodsample in the sample channel and leading electrolyte (or separation)channel 2703 from the elution channel and reservoir 2704, before andafter ITP purification of 50% by volume whole blood lysate 2702. Thepurified nucleic acid is stained with green dye for visualization inelution well. FIG. 27C shows the chip before ITP (blood lysate and ITPbuffers loaded in chip; pure buffer and no DNA in elution well). FIG.27D shows the chip after ITP (blood lysate and ITP buffers loaded inchip; pure buffer and DNA in elution well). FIG. 27E shows the chip postITP purification, with a white light image of the chip channel showingphysical separation of heme from the blood sample in the sample well2705 and leading electrolyte (or separation) channel from the elutionchannel and reservoir 2706 in single channel chip device (50% by volumeblood). FIG. 27F shows the chip post ITP purification, with a whitelight image of the chip channel showing physical separation of heme fromthe blood sample in the sample well 2705 and leading electrolyte (orseparation) channel from the elution channel and reservoir 2706 insingle channel chip device (25% by volume blood).

Example 10—Extraction of High Molecular Weight DNA from Cultured HumanCancer Cells Using Off-Chip Lysis and On-Chip ITP Purification

FIG. 28 shows results of high molecular weight DNA purification for ITP(solid line) compared to solid phase extraction (SPE; dashed line,Qiagen MagAttract) of cultured human Jurkat cells as the percentage ofDNA mass in the purified sample having fragments shorter than a givenlength (Kb). Cell lysis was performed off-chip in a buffered lysissolution containing 10 mM Tris with 5.6 mM HCl and 0.2% v/v IGEPALCA-630. The buffered solution was configured to lyse the cells andreduce mechanical disruption of the DNA during lysis. Cell pellets werelysed in the lysis solution and mixed gently with inversion andslow-speed (automated pipettor), wide-bore tip pipetting (e.g. Rainin200 μl wide bore tip) to aid in homogenization of the lysate. A finalconcentration of 500 μg/ml Proteinase K was added to the lysate andincubated for 20 min at 60° C. ITP was performed on the lysate with 88mM Tris with 44 mM HCl as the leading electrolyte and 1.2 M Tris with0.3 M caproic acid and 0.6 M MOPS as the trailing electrolyte. ITP-basedpurification led to 2 to 3 times greater mean DNA fragment lengths ascompared to the bead-based PSE kit, in part due to reduced mechanicalshearing of the DNA during isotachophoresis compared to SPE due to thelack of a solid phase component or high shear forces (e.g. fromcentrifugation) during the extraction process. The ITP purified DNA hadan average DNA length of about 175 Kb (i.e. 50% of the DNA masscontained DNA fragments greater than about 175 Kb) compared to SPEpurification which yielded DNA with an average length of about 75 Kb.More than 60% of the mass of the DNA extracted by ITP contained anaverage fragment length greater than 150 kB. ITP produced at least aboutthree times as many DNA fragments with a size of at least 150 kB thanthe SPE method.

Example 11—Closing of Channels Using Mechanical Member

FIG. 29A shows a fluidic device comprising 8 closed channels. Eachchannel was permanently closed at two locations 2902, 2902 as shown inFIG. 12B by applying a temperature of 150° C. and a pressure of 30pounds (across all 16 locations; i.e. 1.875 pounds per tooth) to thedevice for 1 second with the comb-like mechanical member of FIG. 12A.FIG. 29B shows a zoomed in microscopic view the second channel closurelocation 2902 adjacent the elution reservoir 2903 of each of thechannels. FIG. 29C shows the percent closure calculated as a function offorce applied to the chips. The extent of channel closure was assessedwithout fluid loaded into the channel. Closure was measured by applyinga constant pressure and measuring air flow rate through the channel.Five chips were assessed. Diamonds indicate closure data obtained fromthe first close location 2901 and triangles indicate closure dataobtained from the second close location 2902. Without a force applied,the channels were open or mostly open. A force of 10 pounds across thedevice was sufficient close most of the channels while a force of 30pounds across the device closed all or nearly all of the channelsrepeatedly. FIG. 29D shows the results of conductivity measurements witha conductivity meter to determine if channels are closed. The chipreservoirs and channels were loaded with ITP buffers as described inFIGS. 12A-12D or FIG. 15 (leading electrolyte, a high concentration ofleading electrolyte for buffering, trailing electrolyte, elution buffer,a high concentration of elution buffer for buffering, and sample bufferwithout a biological sample). The channels were closed using themechanical member and then the fluid in the elution reservoir 2903 waspipetted out of the reservoir and collected. The conductivity of theelution fluid was measured and compared to measurement of theconductivity of original (pre-loaded) elution buffer (same bufferinitially loaded in the chip). It was expected that the conductivity ofmeasured fluid would be the same as the original elution buffer ifchannel closure was sufficient to provide fluidic resistance at thefirst channel close location 2901 between the elution reservoir 2903 andthe channel and at the second channel close location 2902 within thechannel connecting the elution reservoir 2903 to the elution bufferingreservoir (via the elution buffering channel; not shown). For a fullyclosed channel, the conductivity of the eluted volume (withoutperforming ITP) can be equal to the conductivity of the elution bufferalone, indicating no transfer of fluids or ions during collection. Foursituations were tested—without a closer (fully open channels), with thefirst close location 2901 closed (partially closed), with both locations2901, 2902 closed (fully closed), or with every reservoir but theelution reservoir sealed with a film seal. In the film seal situation,the channels were not physically deformed by the mechanical member butwere instead sealed with a film applied by the operator in order toincrease resistance to fluid flow. Elution volumes from partially closedchannels showed increased conductivity compared to fully closedchannels. Sealing of the non-elution reservoirs with the film seal wereincreased compared to fully closed channels as well but remainedgenerally less than the conductivity of a 2× elution buffer. Theseconductivity levels were, however, much lower than those obtained fromeluates without channel closure.

FIG. 85 shows contrast images from fluorescence-based imaging of afluorescently-dyed analyte material 8501 in the channel before (leftside—analyte material 8502 clearly visible in 8 chip elution reservoirs)and after (right side—analyte material 8502 clearly less visible, asindicated by reflection 8503 in 8 individual chip elution reservoirs)pipetting out of the elution well following channel closing with thetooth-like member structure and mechanical actuator for closing thechannels. The fluorescently-dyed analyte material 8501 in the channeldid not move during the elution process, indicating that the channelcloser prevented flow during the elution process.

FIG. 86 shows contrast images from fluorescence-based imaging of afluorescently-dyed analyte material 8601 in the channel before (leftside—analyte material 8602 clearly visible in 8 chip elution reservoirs)and after (right side—analyte material 8602 clearly less visible in 8individual chip elution reservoirs, indicating it was removed) pipettingout of the elution well following channel closing with the PCR filmmembrane seal approach for closing the channels. The fluorescently-dyedanalyte material 8601 in the channel did not move during the elutionprocess, indicating that the channel closer prevented flow during theelution process.

Example 12—Closing of Channels Using Ridge-Like Mechanical Member

FIG. 87A shows conductivity data that was obtained by using aconductivity meter to measure the conductivity of eluted material(namely the conductivity of the ions contained in the eluted liquidvolume) pipetted out of the elution reservoir from a chip. Thenon-elution wells were sealed using either a disposable PCR film (i.e. amembrane-enabled top seal to fluidic reservoirs of chip) or a ridge-likechannel closer as shown in FIG. 54A. Sealing on the non-elution wellsresulted in partial closure of the wells and higher conductivity thanthat of a 2× elution buffer. The conductivity levels of the eluatecollected from the chip sealed with the ridge channel closer waslower-still compared to the PCR film seal, indicating a full ornear-full closure of the channels had been attained. FIG. 87B showscontrast images from fluorescence-based imaging of a fluorescently-dyedanalyte material 8701 in the elution reservoirs before (leftside—material clearly visible in 8 chip elution reservoirs) and after(right side—material clearly less visible in 8 individual chip elutionreservoirs) pipetting out of the elution well following channel closingwith the ridge-like member structure and mechanical actuator for closingthe channels.

Example 13—Closing of Channels Using Channel Closer with Rubber SealingMember and Mechanical Actuator

Table 2 shows conductivity data collected for eluted material recoveredfrom a fluidic device channel closed with the device described in FIG.58A. Each of the channels on an 8-channel fluidic chip were loaded withITP buffers (5 buffer wells per channel) and sample (1 sample well perchannel). The elution buffer was 10 mM Tris HCl pH 7.5 with 0.002% Tween20. The channels were closed by applying a channel closer with a rubbersealing member and a mechanical actuator. A manual pipet was used toremove 50 ul of material from the 8 elution reservoirs followingcompletion of ITP. The conductivity of each eluate was measured with aconductivity meter and compared to the conductivity of pure(unused/unloaded) elution buffer. It was expected that the conductivityof measured fluid would be the same as the original elution buffer ifchannel closure was sufficient to provide fluidic resistance at thechannel closure locations. A partially-closed channel was expected toresult in a conductivity ratio (measured/pure buffer) of between about 1and about 2, with a preferred conductivity ratio value being within arange of about 1 to about 1.5. Channel closure with the rubber sealingmember and mechanical actuator resulted in full closure in 6 of the 8channels and partial closure in the other 2 channels.

TABLE 2 Channel Load Conductivity Ratio number (g) (Eluted material/1×elution buffer) 1 1324 1.2 2 1324 1 3 1324 1 4 1324 1 5 1324 1 6 1324 17 1324 1 8 1324 1.2

Example 14—Voltage Measurement and End-of-Run Triggering

FIG. 30 shows an exemplary example using measurement of the drivingvoltage to trigger a reduction or removal of an electric current in oneof the channels. A fluidic device comprising 8 channels was loaded withITP buffers (leading electrolyte buffer comprising 88 mM Tris with 44 mMHCl, a high concentration of leading electrolyte for buffering, trailingelectrolyte buffer comprising 1.2 M Tris with 0.3 M caproic acid and 0.6M MOPS, an elution buffer comprising 10 mM Tris with 5.6 mM HCl, a highconcentration of elution buffer for buffering) in each of the channels.A sample comprising 50,000 immortalized human cells lysed using themethods described herein was prepared and loaded into each of thechannels. A pre-elution isotachophoresis separation was performed bydriving 900 μA per channel through the channel for 1900 seconds. After1900 seconds 3001, the current was reduced and 250 μA was applied toeach channel to drive the nucleic acids into the elution reservoir. 100seconds after starting isotachophoresis, signal processing using thevoltage on the driving electrode as the data source was started 3002.The top line shown represents the voltage and the bottom line representsthe derivative of the voltage. Two triggers were used to change thedriving current (corresponding to triggers 1 and 4 described in FIG. 16,at locations C and D, respectively). Low-conductivity ions (e.g. sampleions or trailing electrolytes) entering the elution reservoir or channelcan be detected by monitoring for peaks or maximums in the derivative ofthe voltage. The current was turned on at a first trigger point(trigger 1) 3001 to direct nucleic acids into the channel comprisingelution buffer and signal processing 3002 was started shortlythereafter. A first increase was detected at trigger point 3 3003 as thenucleic acids entered the elution reservoir and a second increase wasdetected at trigger point 4 3004 as the trailing electrolytes began toenter the elution reservoir. The current was removed following detectionof the second increase at trigger point 4 3004 so as to position orisolate the sample nucleic acids in the elution well.

Example 15—Temperature Sensing and End-of-Run Triggering

FIG. 21 shows exemplary temperature measurement results using aninfra-red thermal sensor to trigger a reduction or elimination of anelectric current in one of the channels. A fluidic device comprising 8channels was loaded with ITP buffers (leading electrolyte buffercomprising 88 mM Tris with 44 mM HCl, a high concentration of leadingelectrolyte for buffering, trailing electrolyte buffer comprising 1.2 MTris with 0.3 M caproic acid and 0.6 M MOPS, an elution buffercomprising 10 mM Tris with 5.6 mM HCl, a high concentration of elutionbuffer for buffering) in each of the channels. A sample comprising50,000 immortalized human cells lysed using the methods described hereinwas prepared and loaded into each of the channels. A pre-elutionisotachophoresis separation was performed by driving 900 μA per channelthrough the channel for 1900 seconds. After 1900 seconds, the currentwas reduced and 250 μA was applied to each channel to drive the nucleicacids into the elution reservoir. 100 seconds after startingisotachophoresis, signal processing using temperature data collected bya TMP007 infrared temperature sensor. The temperature was detected atlocation 2105 in the elution channel near the elution reservoir 2106,centered approximately 4.5 mm from the elution well 2106. Thetemperature sensor was place approximately 1 mm to 3 mm below the bottomsurface of the fluidic channel. The temperature sensor may be centeredapproximately 4.5 mm from the elution reservoir 2106 (with edges atabout 3.55 mm to about 5.45 mm from the elution reservoir). Thetemperature sensor was configured to detect temperature changes due toelectrophoretic Joule heating in the channel. Electrophoretic joule heatdissipation per channel volume may be inversely proportional toconductivity at a constant current. During isotachophoresis,lower-conductivity ions (trailing electrolytes) may displace higherconductivity ions (leading electrolytes) as the ITP zone moves throughthe channel. The temperature sensor may sense the ITP zone moving pastthe detection location 2105, and the displacement of ions as the ITPzone moves, as a rise in temperature within the channel at the detectionlocation.

The top line shows the temperature at the detection location 2105 andthe bottom line shows the derivative of the temperature. The temperaturewas monitored in real-time for high derivatives in order to detectlower-conductivity buffer zones. The vertical lines indicate when keyevents occurred during monitoring. From left to right, the first line2101 indicates the time at which the current was turned on and thesecond line 2102 indicates the start of signal processing shortlythereafter. The third line 2103 indicates the first detection of anincrease in the derivative of the temperature, and the fourth line 2104indicates the second detection of an increase in the derivative of thetemperature, at which point the current was stopped and the voltage wasdisabled so as to land the voltage in the reservoir and position orisolate the nucleic acids in the elution reservoir.

Example 16—Temperature Sensing and End-of-Run Triggering

FIGS. 88A-88F show exemplary temperature measurement results using aninfra-red thermal sensor to trigger a reduction or elimination of anelectric current in one of the channels. FIG. 88A shows the temperaturewithin the channel as monitored over time by an infrared sensorpositioned as in FIG. 21. The vertical lines indicate when key eventsoccurred during monitoring. Two temperature rises 8801 and 8802 wereused to determine when to begin signal processing and when to end therun, respectively, as described in FIG. 21. FIG. 88B shows thefirst-order derivative values of the temperature data of FIG. 88A. Thetwo peaks of first derivatives 8803 and 8804 correspond to events 8801and 8802 in FIG. 88A. The identification of these two events was basedon a peak detection algorithm. To evaluate the first-order derivatives,temperature data were collected as consecutive time groups within a timewindow. Within each time window, data were fitted by a low orderpolynomial function. The first-order derivatives were then calculatedfrom the polynomial fit. To suppress noise in this signal, a generallikelihood ratio test (GLRT) was used. The data was also fit to a nullhypothesis of constant temperature. FIG. 88C shows the residual of thedata minus the fit 8806 and the data minus the null hypothesis 8805,which were compared to produce a likelihood ratio. A function of thisratio 8807 was used to scale the derivative. The result was that, inregions in which the null hypothesis had a residual comparable to theline of best fit, the derivative was reduced to near zero. In regionswhere the null hypothesis was a poor fit compared to the best fit, thederivative was maintained. In order to detect the peak of first-orderderivatives, an algorithm was used to track the maximum value of thederivative. When the current value declined below the pre-determinedmaximum value by a certain percentage, a peak was detected/recorded. Thederivative peak value was configured to meet a pre-determined thresholdin order to eliminate the possibilities of noise peaks being detected.The processed data were then fed to the triggering algorithm whichactively searched for the occurrences of the two first-order derivativepeaks based on the peak detection method described above. Once two peakswere successfully detected, the extraction process ended and thetriggering algorithm stopped. As will be understood by one of ordinaryskill in the art, voltage may be detected in addition to or as analternative to the temperature using methods and algorithms similar tothose described herein with regards to temperature alone.

FIG. 88D shows a block diagram of triggering process used. The nucleicacid extraction process began with a separation step (Step 8811). Theinstrument was configured to wait for a pre-determined amount of time(Step 8812) before triggering an elution step (Step 8813). Theinstrument was then instructed by the algorithm to wait for a secondperiod of time (Step 8814) before starting signal processing to searchfor the first derivative peak of IR temperature signals (Step 8815).Once the first derivative peak was found, the algorithm began the searchfor the second derivative peak (Step 8816). Once the second derivativepeak was detected, triggering was finished and the elution step wascompleted by ceasing voltage/current flow within the channel (Step8817). The algorithm was configured to time out if no first or secondderivative peak was detected (Steps 8818 and 8819).

FIG. 88E shows a successful triggering of the nucleic extractionprocess. An ITP concentrated band of nucleic acid 8822 was stopped bythe triggering algorithm in elution reservoir 8821. The temperaturetrace and voltage trace both showed the typical two-step rises (8823 and8824) near the end of the process as expected. FIG. 88F shows a failedtriggering run. The majority of nucleic acid 8825 moved past the desiredstop location (elution reservoir 8826) and before being stopped in areservoir further downstream (on the right). The correspondingtemperature and voltage traces showed no typical two-step rises. Onlyone rise step was properly presented in each trace. The triggeringalgorithm timed out after the first derivative peak was found and failedto trigger an end to the run, thereby resulting in the nucleic acid 8825overshooting its target reservoir 8826.

Although a temperature trace was used in this example, the triggeringalgorithm may also rely on a voltage trace solely or voltage andtemperature traces together to perform triggering.

Example 17—Slow Mobility Ion in Sample for Improved Passage Through aCapillary Barrier

FIG. 89A shows a capillary barrier 8901 between sample (in this case,sample prepared in leading electrolyte) and leading electrolyte buffer,for example as shown in FIG. 10A. In some instances, such capillarybarriers 8901 can trap nucleic acid during an (ITP-based) extractionprocess. To facilitate the passage of the nucleic acid past such acapillary barrier 8901, an ion with a slow magnitude of mobility wasadded into sample to disturb the accumulation of nucleic acids (e.g.into a tightly focused ITP band prior to reaching the capillary barrier8901 which may impede passage of the band). This disturbance resulted inchanges in the morphology of the nucleic acids and enabled the passageof the nucleic acids past the capillary barrier 8901 without loss of thenucleic acid sample. FIG. 89B compares the passage of a nucleic acidsample with the addition of a slow ion (14 mM3-(N-morpholino)propanesulfonic acid (MOPS)) 8902 versus a control 8903(i.e., same sample without the addition of a slow ion). The sample withslow ions passed the barrier successfully. In contrast, the controlchannel 8903 showed nucleic acid 8904 (stained so that it may bevisualized in the channel during ITP) trapped at capillary barrier 8901.FIG. 89C demonstrates the nucleic acid morphology sometime later in theextraction process. Because of the trap of the nucleic acids caused bythe capillary barrier in the control channel, the control sample 8903comprises a longer “tail” 8905 of nucleic acids in the channel comparedto the sample with added MOPS 8902, which may lead to loss of nucleicacids further downstream if a distinct ITP band does not form (or ifsample remains captured near the capillary barrier) by the time the ITPprocess is stopped and the sample is eluted from the elution well asdescribed herein.

Example 18—Shifted Liquid-Liquid Interface for Improved Passage Througha Capillary Barrier

FIG. 89D shows the capillary barrier 8911 between leading electrolytebuffer and elution buffer. In some instances, capillary barrier 8911 cantrap nucleic acid (NA) during the elution process (during ITP). Tofacilitate the passage, we adjusted the position of the fluid-to-fluidinterface between the leading electrolyte buffer and the elution bufferby adjusting the liquid head heights in reservoirs as described in FIGS.43 and 44. FIG. 89E shows examples of adjusting buffer interfaces 8912.We spiked in a fluorescence dye in elution buffer to visualize theinterface. The upper lane 8910 shows an instance where the interface8912 was moved to the right of barrier 8911, away from elution reservoir8913. The lower lane 8920 shows an instance where the interface 8912 wasmoved to the left of the barrier 8911, toward elution reservoir 8913.When nucleic acid transitioned from leading electrolyte buffer toelution buffer, the volume of nucleic acid expanded to adapt to thechange of ionic strength as described herein. When this expansionoccurred before the passage of capillary barrier 8912 (e.g. at thelocation indicated in 8910), some nucleic was be trapped due to thelimit of volume that the capillary barrier can allow to pass. However,when this expansion occurred after the passage of the capillary barrier8912 (e.g. at the location indicated in 8920), NA can stay in compactshape and pass through without any loss. FIG. 89F shows how nucleic acidpassage over the capillary barrier 8911 was facilitated or hampered byadjusting interface 8912 position. The upper lane 8910 had the bufferinterface 8912 on the right of the capillary barrier 8911 and the lowerlane 8920 had the interface 8912 on the left of the capillary barrier8911. Some nucleic was trapped at the capillary barrier 8911 in upperlane 8910, whereas no nucleic was observed at the barrier 8911 in thelower lane 8920 but was instead found in the elution reservoir 8913.

Example 19—Simultaneous ITP in an 8-Channel Fluidic Device

FIG. 31A, FIG. 31B, FIG. 31C, and FIG. 31D show the results ofsimultaneously performing isotachophoresis in 8 channels of a fluidicdevice. DNA was extracted from cell culture lysate using a bench topcontroller device to automate isotachophoresis in a monolithic,8-channel fluidic device. Leading electrolyte buffer containing 88 mMTris with 44 mM HCl and 0.002% Tween 20 was loaded into the leadingelectrolyte reservoir of each channel. Trailing electrolyte comprising1.2M Tris with 0.3 M Caproic Acid and 0.6 M MOPS with 0.002% Tween 20was loaded into the trailing electrolyte reservoir of each channel. Thesample for each channel was prepared in a second leading electrolytebuffer (e.g. sample buffer) comprising 10 mM Tris with 5.6 mM HCl with0.002% Tween 20. Each sample comprised a cell lysate. The total numberof human COLO 320 ccells per sample represented 100,000 diploid genomeequivalents. Cells were pelleted and lysed off-chip in a lysis solutioncomprising 40 mM NaOH for 2 minutes and subsequently quenched at a 1:1volume ratio with a buffered acidic solution to bring the final celllysate sample to 10 mM Tris with 5.6 mM HCl and 20 mM NaCl at pH 8.Proteinase K was added to a final concentration of 400 μg/ml within thecell lysate sample volume. Four of the eight samples were treated withRNase A at a final concentration of 200 μg/ml and allowed to stand atroom temperature for 2 minutes. All eight samples were then incubatedfor 10 minutes at 56° C. The lysed samples were then brought to roomtemperature and loaded into the separate eight samplereservoirs/channels on the microfluidic device, denoted by channels Athrough H, in preparation for isotachophoresis. The four samples thatwere not treated with RNase A were loaded into channels B, D, F and H.The four samples treated with RNase A were loaded into channels A, C, E,and G. Samples treated with RNase A contained additional buffering ionsto enable optimal RNase activity, and therefore represented higher ionicstrength or higher conductivity samples that, under fixed current (ITPconditions), resulted in different voltage data traces than the samplesnot treated with RNase. The independent electrical circuit control ofchannels A through H enabled voltage signal and feedback control forautomated control and end-run triggering for each of the differentchannels of the device. FIG. 31A shows a micrograph of ITP bands withfocused DNA 3101 in each of the 8 samples in the sample channel regionof the device. The ITP band of DNA 3101 migrates within the channel inresponse to an applied electric field. The ITP band first travels awayfrom the trailing electrolyte reservoir in the direction indicated byarrow 3102. The ITP band 3101 then traverses the 180° low-dispersionturn and continues through the channel towards the elution reservoir inthe opposite direction (with respect to the chip) 3103 in response tothe applied electric field. FIG. 31B shows independent voltage signaldata at fixed currents for each of the 8 channels over time. FIG. 31Cshows a micrograph of the same 8 ITP bands with focused DNA 3101 fromthe samples eluted in the elution reservoir by independently controlledend-of-run voltage based triggering (this image represents the end ofthe run with the electrical field automatically shut off). FIG. 31D is amagnified section of the voltage tracing (monitoring) used fortriggering shown in FIG. 31B. The electric current of each channel wasindependently applied to the channel and the voltage of each channel wasindependently monitored in order to trigger a change (in this casecessation) in the electric field applied to each channel independentlyof every other channel.

Example 20—Off-Chip Lysis Efficiency

FIGS. 90A-90C show the lysis efficiency of three different cells lineslysed as described in FIG. 32A. FIG. 90A shows the lysis efficiency ofCOLO320 cells. The COLO320 cells were cultured in a mixture of suspendedand adherent cells. FIG. 90B shows the lysis efficiency of GM24385cells. The GM24385 cells were cultured in suspension. FIG. 90C shows thelysis efficiency of H2228 cells. The H2228 cells were cultured asadherent cells. In each of FIGS. 90A-90C, the cells were harvest andtitrating amounts of cell inputs were lysed as described herein. FIGS.90A-90C show scatterplots relating theoretical yield based on input cellnumber to observed yield as measured by the Qubit dsDNA assay. Cellinputs were corrected for ploidy (COLO320=2.27N; GM24385=2N1 H222=3.74N)and the theoretical yield was calculated based on an assumption of 6.6pg of DNA per cell. FIG. 90A shows an R² value of 0.9407 for the COLO320cells, FIG. 90B shows an R² value of 0.99997 for the GM24385 cells, andFIG. 90C shows an R² value of 0.98917 for the H2228 cells, indicating astrong linear relationship between cell input and yield (prior to ITP)across a range of cell types and sample amounts.

Example 21—Temperature Sensing and End-of-Run Triggering

FIGS. 91A-91B show exemplary temperature measurement results using aninfra-red thermal sensor to trigger a reduction or elimination of anelectric current in one of the channels. The chip was an 8-channel chipsubstantially similar to that shown in FIG. 38. FIG. 91A shows the ITPband 9101 successfully stopped at the end of the ITP run in a tight bandwithin the elution reservoir 9102. FIG. 91B shows the temperature withineach channel as monitored over time by an infrared sensor positioned asin FIG. 40. Arrow 9103 indicates the time at which the ITP band 9101reached the infrared sensor location.

Example 22—Voltage and Temperature Sensing for End-of-Run Triggering

FIG. 92A shows a block diagram of sample channel to LE channeltriggering process used. The nucleic acid extraction process began witha separation step (Step 9201). The instrument was then instructed by thealgorithm to wait for a first period of time (Step 9202) before startingsignal processing to search for the first derivative peak of the voltagesignal at the capillary barrier between the sample channel and the LEchannel (Step 9202). Once the first derivative peak was found (Step9203), the algorithm began the search for the second derivative peak ofthe voltage signal at the channel narrowing after the capillary barrierwithin the LE channel. Alternatively, if no first peak derivative of thevoltage signal was found, the instrument would time out after a secondperiod of time (Step 9205) and the algorithm would begin searching forthe second derivative peak of the voltage signal (Steps 9204, 9211).

FIG. 92B shows a trace of the voltage 9206, the derivative of thevoltage 9207, and the measurement error 9208 at the first triggeringlocation at the capillary barrier between the sample channel and the LEchannel as a function of time.

FIG. 92C shows a block diagram of LE channel triggering process used.The instrument was instructed to search for the second derivative peak(Step 9204, 9211). The instrument was instructed by the algorithm tostart signal processing to search for the second derivative peak at thenarrowing after the capillary barrier within the LE channel (as shown inFIG. 40) (Step 9212). Once the second derivative peak was detected, theinstrument was instructed to start searching for the first derivativepeak of the temperature with the IR sensor within the elution branch ofthe channel (Step 9213, 9221). Alternatively, if no second peakderivative of the voltage signal was found, the instrument would timeout after a third period of time (Step 9214) and the algorithm wouldbegin searching for the first derivative peak of the temperature (Steps9213, 9221).

FIG. 92D shows a trace of the voltage 9215, the derivative of thevoltage 9216, and the measurement error 9217 at the second triggeringlocation at the narrowing after the capillary barrier within the LEchannel as a function of time.

FIG. 92E shows a block diagram of the elution triggering process used.The instrument was then instructed by the algorithm to wait for a fourthperiod of time before starting signal processing (Step 9213, 9221) tosearch for the first derivative peak of the temperature signal in theelution branch (Step 9222). Once the first derivative peak was found,the algorithm began the search for the second derivative peak of thetemperature signal in the elution branch (Step 9223). Alternatively, ifno first peak derivative of the temperature was found, the instrumentwould time out after a fifth period of time (Step 9224). Once the secondderivative peak was detected, triggering was finished and the elutionstep was completed by ceasing voltage/current flow within the channel(Step 9225). The algorithm was configured to time out after a sixthperiod of time if no second derivative peak was detected (Step 9226).

FIG. 92F shows a trace of the voltage 9227, the derivative of thevoltage 9228, and the measurement error 9229 at the temperaturetriggering location within the elution branch of the channel.

“A”, “an”, and “the”, as used herein, can include plural referentsunless expressly and unequivocally limited to one referent.

As used herein, the term “or” is used to refer to a nonexclusive or,such as “A or B” includes “A but not B,” “B but not A,” and “A and B,”unless otherwise indicated. As used herein, the term “or” means “and/or”unless stated otherwise.

The term “about” as used herein, unless otherwise indicated, refers to avalue that is no more than 10% above or below the value being modifiedby the term. For example, the term “about −20° C.” means a range of from−22° C. to −18° C. As another example, “about 1 hour” means a range offrom 54 minutes to 66 minutes.

The term “substantially” as used herein, unless otherwise indicated,refers to a value that is no more than 30% above or below the valuebeing modified by the term. For example, the term “substantially −20°C.” means a range of from −26° C. to −14° C.

The term “approximately” as used herein, unless otherwise indicated,refers to a value that is no more than 10% above or below the valuebeing modified by the term. For example, the term “approximately −20°C.” means a range of from −22° C. to −18° C. As another example,“approximately 1 hour” means a range of from 54 minutes to 66 minutes.

The term “substantially flat” as used herein, unless otherwiseindicated, refers to surfaces that have their main extension in oneplane in contrast to being shaped, for example a surface which is atleast 70% linear.

The term “substantially parallel” as used herein, unless otherwiseindicated, refers to a plane that largely extends in the same directionas the plane which is being modified by the term, for example a planethat is no more than 30° off axis from the plane parallel to the valuebeing modified by the term. For example, the term “substantiallyparallel to the surface” means a plane which is within 30° of the planeparallel to the surface.

The term “substantially perpendicular” as used herein, unless otherwiseindicated, refers to a plane that largely extends perpendicularly (i.e.along a plane that is 90° relative) to the plane which is being modifiedby the term, for example a plane that is no more than 30° off axis fromthe plane perpendicular to the value being modified by the term. Forexample, the term “substantially perpendicular to the surface” means aplane which is within 30° of the plane perpendicular to the surface.

The term “relatively aligned with” as used herein, unless otherwiseindicated, refers to a value that large extends in the same direction asthe value which is being modified by the term, for example along an axisthat his not more than 30° off axis from the value being modified by theterm. For example, the term “relatively aligned with the longitudinalaxis” means an extending along an axis which is within 30° of thelongitudinal axis.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It can be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A fluidic device comprising an isotachophoresis(ITP) circuit comprising: a first channel comprising first and secondcapillary barriers that are spaced apart; a first loading reservoir influid communication with said first channel via a first aperture in saidfirst channel, wherein said first aperture is positioned between saidfirst and second capillary barriers to permit a liquid entering saidfirst channel via said first aperture to flow in one direction alongsaid first channel and arrest at said first capillary barrier and toflow in another direction along said first channel and arrest at saidsecond capillary barrier.
 2. The fluidic device of claim 1, wherein saidliquid entering said first channel via said aperture flows along a pathto said first or second capillary barrier that is longer than a width ofsaid first channel.
 3. The fluidic device of claim 1, wherein saidliquid entering said first channel via said aperture flows such that ameniscus of said first liquid arrests at said first capillary barrier orat said second capillary barrier, or at both said first and said secondcapillary barrier.
 4. The fluidic device of claim 1, wherein said firstcapillary barrier is configured and arranged to be breached by a liquidwhen a first burst pressure is applied to said one or more branchedfluidic circuits and said second capillary barrier is configured andarranged to be breached by said liquid when a second burst pressure isapplied to said one or more branched fluidic circuits, wherein saidfirst and said second burst pressures are about equal or wherein saidfirst burst pressure is greater than said second burst pressure.
 5. Thefluidic device of claim 1, wherein one or both of said first and secondcapillary barriers is a cliff capillary barrier or a plateau capillarybarrier.
 6. The fluidic device of claim 5, wherein said plateaucapillary barrier is configured and arranged so that an air gap formsbetween said first liquid after said first liquid arrests at saidplateau capillary barrier and a second liquid after said second liquidflows toward said plateau capillary barrier in another direction andarrests at said plateau capillary barrier opposite to said first liquid.7. The fluidic device of claim 1, wherein said ITP circuit comprises asecond channel in fluid communication with said first channel and saidfirst capillary barrier is configured and arranged to arrest flow of asecond liquid as it flows along said second channel such that aliquid-liquid interface is formed between said first and second liquidsat said first capillary barrier.
 8. The fluidic device of claim 1,wherein said ITP circuit further comprises a second loading reservoirand a second channel, wherein said second loading reservoir is in fluidcommunication with said second channel via a second aperture and saidsecond channel comprises a third capillary barrier wherein said thirdcapillary barrier is configured and arranged to use capillary forces toarrest a meniscus of a liquid flowing along said second channel at saidthird capillary barrier.
 9. The fluidic device of claim 8, wherein saidITP circuit further comprises a third loading reservoir fluidlyconnected to a third channel via a third aperture, wherein said thirdchannel is fluidly connected to said second reservoir, wherein saidthird channel comprises a fourth capillary barrier positioned betweensaid second aperture and said third aperture.
 10. The fluidic device ofclaim 1, wherein said ITP circuit comprises an elution channel connectedto a first elution reservoir at an elution junction.
 11. The fluidicdevice of claim 1, wherein said first capillary barrier or said secondcapillary barrier, or both, is adjacent to an air channel comprising aconstriction.
 12. The fluidic device of claim 1, further comprising oneor more pneumatic channels opening at one or more pneumatic ports and incommunication with each of said capillary barriers.
 13. The fluidicdevice of the preceding claim, further comprising: (a) a substratehaving a first face and a second face, wherein said first face comprisesa plurality of reservoirs including said first loading reservoir andsaid one or more pneumatic ports and said second face comprises aplurality of channels including said first channel, wherein saidplurality of reservoirs communicate with said plurality of channels viathrough holes in said substrate; (b) a layer of material covering saidsecond face, thereby forming closed channels; and (c) a cover coveringat least part of said first face and comprising through holes thatcommunicate with ports in said first face through gaskets.
 14. Thefluidic device of claim 13, wherein said one or more pneumatic portshave a head height relative to said substrate that is shorter than saidfirst loading reservoir.
 15. The fluidic device of claim 13, whereinsaid cover further comprises a porous, air-permeable, hydrophobicmaterial positioned between the through holes in the ports.
 16. Afluidic device comprising a fluidic channel and disposed in said fluidicchannel a capillary barrier that restricts flow of a liquid in saidfluidic channel, wherein said capillary barrier comprises: a rampprotruding from a surface of said fluidic channel at a first angle; aplateau area; and a cliff area extending from said plateau area to saidsurface of said fluidic channel and wherein said cliff area intersectswith said surface at a second angle that is substantially steeper thansaid first angle.
 17. A fluidic device comprising a fluidic channel anddisposed in said fluidic channel a capillary barrier that restricts flowof a liquid in said fluidic channel, wherein said capillary barriercomprises: (a) a first ramp protruding from a surface of said fluidicchannel at a first angle that is less than 80 degrees; (b) a plateauarea; and (c) a second ramp extending from said plateau area to saidsurface of said fluidic channel and wherein said second ramp intersectswith said surface at a second angle that is less than 80 degrees.
 18. Afluidic device comprising one or more branched fluidic circuits, whereineach of said branched fluidic circuits comprises an isotachophoresis(“ITP”) branch and an elution branch in communication with said ITPbranch, wherein: (a) said ITP branch comprises a trailing electrolytebuffer reservoir, a sample channel, a leading electrolyte bufferchannel, a first leading buffer electrolyte reservoir and a secondleading electrolyte buffer reservoir, all in communication with eachother, wherein: (i) said sample channel is separated from said trailingelectrolyte reservoir by a first cliff capillary barrier and from saidleading electrolyte buffer channel by a second cliff capillary barrier,(ii) said leading electrolyte reservoir is separated from said secondleading electrolyte reservoir by a first plateau capillary barrier; and(b) said elution branch comprises an elution channel, a first elutionbuffer reservoir and a second elution buffer reservoir, all incommunication with each other, wherein: (i) said first elution bufferreservoir is separated from said second elution buffer reservoir asecond plateau capillary barrier, and (ii) said leading electrolytebuffer channel is separated from at least part of said elution channelby a third plateau capillary barrier.
 19. A device for performingvertical isotachophoresis, the device comprising: one or morecylindrical columns comprising an interior channel defined by an innerwall of the cylindrical column, each cylindrical column comprising: afirst stage comprising a first gel plug disposed at a first locationwithin the interior channel and a first space disposed within theinterior channel between the first gel plug and an upper end of thecylindrical column; a second stage comprising a second gel plug disposedat a second location within the interior channel, the second locationbeing located below the first location and oriented in line withgravity, and a second space disposed within the interior channel betweenthe first gel plug and the second gel plug; and a third stage comprisinga third gel plug disposed at a third location within the interiorchannel, the third location being located below the second location andoriented in line with gravity, and a third space disposed within theinterior channel between the second gel plug and the third gel plug.